Therapeutic agents—I

ABSTRACT

The present invention relates generally to chemical agents useful in the treatment and prophylaxis of infection by pathogenic or potentially pathogenic entities, or entities capable of opportunistic infection in mammals, including humans and primates, non-mammalian animals and avian species. More particularly, the present invention provides a chemical agent of the macrocyclic diterpene family obtainable from a member of the Euphorbiaceae family of plants or botanical or horticultural relatives thereof or derivatives or chemical analogues or chemically synthetic forms of the agents for use in the treatment or prophylaxis of infection by pathogenic entities in mammalian, animal and avian subjects. The present invention further contemplates a method for the prophylaxis and/or treatment in mammalian, animal or avian subjects of infection or potential infection by pathogenic entities by the topical or systemic administration of a macrocyclic diterpene obtainable from a member of the Euphorbiaceae family of plants or their botanical or horticultural derivatives or a derivative, chemical analogue or chemically synthetic form of the agent. The chemical agent of the present invention may be in the form of a purified compound, mixture of compounds, a precursor form of one or more of the compounds capable of chemical transformation into a therapeutically active agent or in the form of a chemical fraction, sub-fraction, preparation or extract of the plant.

CROSS REFERENCE TO RELATED APPLICATION

This is a continuation in part of copending PCT applicationPCT/AU01/00679, filed on Jun. 7, 2001.

FIELD OF THE INVENTION

The present invention relates generally to chemical agents useful in thetreatment and prophylaxis of infection by pathogenic or potentiallypathogenic entities, or entities capable of opportunistic infection inmammals, including humans and primates, non-mammalian animals and avianspecies. More particularly, the present invention provides a chemicalagent of the macrocyclic diterpene family obtainable from a member ofthe Euphorbiaceae family of plants or botanical or horticulturalrelatives thereof or derivatives or chemical analogues or chemicallysynthetic forms of the agents for use in the treatment or prophylaxis ofinfection by pathogenic entities in mammalian, animal and aviansubjects. The present invention further contemplates a method for theprophylaxis and/or treatment in mammalian, animal or avian subjects ofinfection or potential infection by pathogenic entities by the topicalor systemic administration of a macrocyclic diterpene obtainable from amember of the Euphorbiaceae family of plants or their botanical orhorticultural derivatives or a derivative, chemical analogue orchemically synthetic form of the agent. The chemical agent of thepresent invention may be in the form of a purified compound, mixture ofcompounds, a precursor form of one or more of the compounds capable ofchemical transformation into a therapeutically active agent or in theform of a chemical fraction, sub-fraction, preparation or extract of theplant.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in thisspecification are collected at the end of the description.

Reference to any prior art in this specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in Australia or any othercountry.

Natural product screening is a term applied to the screening of naturalenvironments for bioactive molecules. Particularly sought afterbioactive molecules are those having potential as useful therapeuticagents. Natural environments include plants, microorganisms, coral andmarine animals. The search for potential therapeutic agents for thetreatment of cancer and infection by pathogenic organisms remains animportant focus.

The Euphorbiaceae family of plants covers a wide variety of plantsincluding weeds of Euphorbia species. There have been a variety ofinconclusive reports on the potential effects of the sap of these plantson a variety of conditions as well as promoting tumorigenesis andcausing skin and ocular irritation.

The most intensively studied species of this group is Euphorbiapilulifera L (synonyms E. hirta L., E. capitata Lam.), whose commonnames include pill-bearing spurge, snakeweed, cat's hair, Queenslandasthma weed and flowery-headed spurge. The plant is widely distributedin tropical countries, including India, and in Northern Australia,including Queensland.

A recent report describes selective cytotoxicity of a number oftiglilane diterpene esters from the latex of Euphorbia poisonii, ahighly toxic plant found in Northern Nigeria, which is used as a gardenpesticide. One of these compounds has a selective cytotoxicity for thehuman kidney carcinoma cell line A-498 more than 10,000 times greaterthan that of adriamycin (Fatope et al., 1996).

Euphorbia hirta plants and extracts thereof have been considered for avariety of purposes, including tumor therapy (EP 0 330 094),AIDS-related complex and AIDS (HU-208790) and increasing immunity and asan anti-fungoid agent for treatment of open wounds (DE-4102054).

Thus, while there are isolated reports of anti-cancer activity ofvarious Euphorbia preparations (see Fatope et al., 1996; Oksuz et al.,1996), not only are the compounds present in at least one Euphorbiaspecies reported to be carcinogenic (Evans and Osman, 1974; Stavric andStolz, 1976; Hecker, 1970), but at least one species has a skin-irritantand tumor-promoting effect (Gundidz et al., 1993) and another speciesreduces EBV-specific cellular immunity in Burkitt's lymphoma (Imai,1994).

In accordance with the present invention, the inventors have identifiedchemical agents and fractions comprising these agents which are usefulin the treatment and prophylaxis of infection by pathogenic orpotentially pathogenic entities, or entities capable of opportunisticinfection in mammals, including humans and primates, non-mammaliananimals and avian species.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

The present invention is predicated in part on the identification ofchemical agents and fractions comprising same from plants of theEuphorbiaceae family which are useful in the treatment and prophylaxisof infection by pathogenic, potentially pathogenic and opportunisticorganisms. Such organisms include prokaryotes, eukaryotes and viruses.The inventors have further identified that the chemical agents of thepresent invention are capable of modulating protein kinase C (PKC)activity thus providing a basis for the treatment of conditions wherePKC activity is required to be up-regulated or down-regulated.

Accordingly, one aspect of the present invention contemplates a methodfor the treatment or prophylaxis of a condition associated with thepresence of a biological entity or part thereof or a toxin or venomtherefrom or a genetic event caused thereby in a subject, said methodcomprising the administration to said subject of a symptom-amelioratingeffective amount of a chemical agent obtainable from a plant of theEuphorbiaceae family or a derivative or chemical analogue thereof whichchemical agent is a macrocyclic diterpene selected from compounds of theingenane, pepluane and jatrophane families and which chemical agent orderivative or chemical analogue as represented by any one of the generalformulae (I)-(V) as defined herein and which chemical agent orderivative or chemical analogue thereof is capable of modulating PKCactivity, PKC-dependent gene expression or PKC enzyme turnover andwherein said chemical agent or its derivatives or chemical analogues isadministered for a time and under conditions sufficient to ameliorateone or more symptoms associated with said biological entity.

Yet another aspect of the present invention contemplates a method forthe treatment or prophylaxis of a microbial infection in a subject, saidmethod comprising the administration to said subject of asymptom-ameliorating effective amount of a macrocyclic diterpene, or achemical fraction comprising same from a plant of the familyEuphorbiaceae or a derivative or chemical analogue of said macrocyclicditerpene having the structures as defined above wherein saidmacrocyclic diterpene or its derivative or chemical analogue modulatesPKC activity, synthesis or enzyme turnover, said administration beingfor a time and under conditions sufficient to ameliorate one or moresymptoms of the infection.

Still another aspect of the present invention provides a method for thetreatment or prophylaxis of an infection by a lower eukaryotic organismin a subject, said method comprising the administration to said subjectof a symptom-ameliorating effective amount of a macrocyclic diterpene orchemical fraction comprising same from a plant of the familyEuphorbiaceae or a derivative or chemical analogue of said macrocyclicditerpene having the structures as defined above wherein saidmacrocyclic diterpene or its derivative or chemical analogue modulatesPKC activity, synthesis or enzyme turnover, said administration beingfor a time and under conditions sufficient to ameliorate one or moresymptoms of the infection.

Still yet another aspect of the present invention provide a method forthe treatment or prophylaxis of an infection by a complex eukaryoticorganism in a subject, said method comprising the administration to saidsubject of a symptom-ameliorating effective amount of a macrocyclicditerpene, or a chemical fraction comprising same from a plant of thefamily Euphorbiaceae or a derivative or chemical analogue of saidmacrocyclic diterpene having the structures as defined above whereinsaid macrocyclic diterpene or its derivative or chemical analoguemodulates PKC activity, synthesis or enzyme turnover, saidadministration being for a time and under conditions sufficient toameliorate one or more symptoms of the infection.

Even yet another aspect of the present invention provide a method forthe treatment or prophylaxis of an infection by a virus in a subject,said method comprising the administration to said subject of asymptom-ameliorating effective amount of a macrocyclic diterpene, or achemical fraction comprising same from a plant of the familyEuphorbiaceae or a derivative or chemical analogue of said macrocyclicditerpene having the structures as defined above wherein saidmacrocyclic diterpene or its derivative or chemical analogue modulatesPKC activity, synthesis or enzyme turnover, said administration beingfor a time and under conditions sufficient to ameliorate one or moresymptoms of the infection.

A further aspect of the present invention contemplates a method ofassessing the suitability of a chemical agent from Euphorbiaceae for thepractice of the present invention. Numerical values are assigned tochemical agents including fractions comprising the chemical agents asset forth, for example, in Table A:

TABLE A Feature Value An ability to modulate PKC activity or effect +1An ability to induce bipolar dendritic activity +1 An ability todisplace phorbol dibutyrate from +1 binding to PKC An ability to inducerespiratory burst in leucocytes +1 An ability to stimulate phagocytosisin peripheral +1 blood mononuclear cells An ability to be derived from amember of the +1 Euphorbiaceae family. Derived from E. peplus +3 Waterextractible from the sap of Euphorbia sp. +2 An ability to activatelatent virus in vitro +4 A lower tumor promotion activity than TPA/PMA+2

Another aspect of the present invention contemplates a method for thetreatment or prophylaxis of infection or colonization or presence of abiological entity in a subject, said method comprising administration tosaid subject of a symptom-ameliorating effective amount of a macrocyclicditerpene obtainable from a Euphorbiaceae plant or its botanical orhorticultural relative, said macrocyclic diterpene being selected froman ingenane, pepluane or jatrophane, or a derivative or chemicalanalogue thereof, having the structure represented by any one of thegeneral formulae (I)-(V) as defined below and wherein said chemicalagent exhibits a potency of agent (P_(A)) of >10, wherein theP_(A)=ΣI_(V) where I_(V) is a numerical value associated with aparticular feature as defined in Table A or pharmaceutically acceptablesalts of these, said chemical agent being administered for a time andunder conditions sufficient to ameliorate at least one symptom caused byor associated with the biological entity.

Yet another aspect of the present invention contemplates a computerprogram product for assessing the likely usefulness of a candidatecompound or group of compounds for treating or preventing infection orcolonization or presence of a biological entity in a subject, saidproduct comprising:

-   (1) code that receives as input index values for at least two    features associated with said compound(s), wherein said features are    selected from:    -   (a) the ability to modulate PKC activity or effect;    -   (b) the ability to induce bipolar dendritic activity;    -   (c) the ability to be derived from a member of the Euphorbiaceae        family;    -   (d) the ability to be derived from E. peplus;    -   (e) the ability to be water extractable from the sap of a        Euphorbia species; or    -   (f) the ability to activate latent virus;    -   (g) less tumor promoting capacity than TPA or PMA;-   (2) code that adds said index values to provide a sum corresponding    to a potency value for said compound(s); and-   (3) a computer readable medium that stores the codes.

Still another aspect of the present invention extends to a computer forassessing the likely usefulness of a candidate compound or group ofcompounds for treating or preventing infection or colonization orpresence of a biological entity in a subject, wherein said computercomprises:

-   (1) a machine-readable data storage medium comprising a data storage    material encoded with machine-readable data, wherein said    machine-readable data comprise index values for at least two    features associated with said compound(s), wherein said features are    selected from:    -   (a) the ability to modulate PKC activity or effect;    -   (b) the ability to induce bipolar dendritic activity;    -   (c) the ability to be derived from a member of the Euphorbiaceae        family;    -   (d) the ability to be derived from E. peplus;    -   (e) the ability to be water extractable from the sap of a        Euphorbia species; or    -   (f) the ability to activate latent virus;    -   (g) less tumor promoting capacity than TPA or PMA;-   (2) a working memory for storing instructions for processing said    machine-readable data;-   (3) a central-processing unit coupled to said working memory and to    said machine-readable data storage medium, for processing said    machine readable data to provide a sum of said index values    corresponding to a potency value for said compound(s); and-   (4) an output hardware coupled to said central processing unit, for    receiving said potency value.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the activation of PKC, using a fluorescent peptide assay(“PepTag” non-radioactive protein kinase kit, Promega). Lane 1, PKC andsubstrate alone; lane 2, plus positive control activator; lane 3, plus100 ng/ml TPA; lane 4, plus 0.1 ng/ml TPA; lane 5, plus 0.01 ng/ml TPA;lane 6, plus 0.001 ng/ml TPA; lane 7, ether extract of E. peplus sap inDEM, diluted 1 in 5; lane 8, aqueous layer from ether extraction,diluted 1/25; lane 9, crude sap diluted 1/25; lane 10, DME alone.

FIG. 2 shows the activation of PKC by E. peplus fractions. Lanes 1 and2, same as FIG. 1; lane 3, 2 mg/ml fraction H; lane 4, 2 mg/mlingenanes.

FIG. 3 is photographic representation showing the results of a PKC assayusing rat brain PKC. Lane 1, negative control; lane 2, positive control;lane 3, empty; lane 4, PEP001 (1/125 dilution), lane 5, PEP001 (1/500dilution) and lane 6, TPA (20 μg).

FIG. 4 is photographic representation showing the activation of PKC inMM96L cells expressing PKC fused to green fluoresent protein (GFP). FIG.4A shows PKCβ expressed in the nuclei of MM96L human melanoma PKC MM96Lcells in the absence of drug. FIG. 4B shows the effect of treatment withcrude E. peplus extract for 2 hr.

FIG. 5 is a photographic representation showing induction oftranslocation of activated PKCs by the compounds of the instantinvention to the cytoplasm, plasma membrane and to the Golgi orsimilarly located cellular structure.

FIG. 6 is a graphical representation showing the induction oftranslation of the classical and novel PKC isoforms in response toPEP003, PEP005, bryostatin-1 and TPA.

FIG. 7 is a graphical representation showing the activation of HIV fromU1 cells.

FIG. 8 is a graphical representation showing treatment of lytic HIVinfection of peripheral blood mononuclear cells (PBMC) with PEP003,PEP004, TPA and ingenol, expressed as p24 production over a 10 daytreatment period. FIG. 8A represents unifected cells. FIG. 8B representslow titer infected cells. FIG. 8C represents lower titer infected cellsrepresented as p24 production versus drug concentration. FIG. 8Drepresents infected cells represented as p24 production versus drugconcentration but at a higher titer infection than FIG. 8C.

FIG. 9 is a photographic representation showing the recruitment ofneutrophils in the skin induced by PEP001 extract. FIG. 9A shows normalskin of nude mouse. FIG. 9B shows skin of nude mouse showinginfiltration of neutrophils one day after treatment with E. peplus sap.

FIG. 10 is a photographic representation showing effect of PEP010 onrecruitment of neutrophils in normal skin of nude mouse and skinoverlaying subcutaneously implanted B16 melanoma. FIG. 10A shows theeffect of 24 hr treatment. FIG. 10B shows the effect of 48 hr treatment.

FIG. 11 is a graphical representation illustrating the ability of PEP001to induce the release of superoxide radical, as demonstrated byfluorescence-activated cell sorting. FIG. 11A depicts a control. FIG.11B depicts the effect of PEP001.

FIG. 12 is a graphical representation showing the effect ofpre-treatment of leukocytes with PEP003 on E. coli activity (16 hrincubation), relative to PBS control; depicted as numbers of E. colicells/ml media.

FIG. 13 is a graphical representation showing the effect ofpre-treatment of leukocytes with PEP003 on E. coli numbers depicted interms of turbidity.

FIG. 14 is a photographic representation showing production of viralcapsid antigen (VCA) in B95-8 (EBV+ Marmoset cell line) after treatmentwith TPA, PEP003 and PEP004 for 3 and 7 days.

FIG. 15 is a photographic representation showing production of viralcapsid antigen (VCA) in BL74 and Mutu I (Burkitts lymphoma cell lines)after treatment with TPA, PEP003 and PEP004 for 3 and 7 days.

FIG. 16 is a photographic representation showing production of BZLF1(the initial transactivator of EBV) after treatment with TPA, PEP003 andPEP004 for 3 and 7 days.

FIG. 17 is a graphical representation showing activation of naturalkiller cell activity, assayed as % specific lysis of K562 cells (anatural killer—sensitive cell line) after pre-treatment of AO2-Mmelanoma cells with PEP003 and TPA.

FIG. 18 is a graphical representation showing survival of Jam cellsafter treatment with saps from various Euphorbiaceae, expressed aspercentage cell survival determined by sulfurhodamine B staining ofcells. FIG. 18A shows the cytotoxicity of the saps alone on the Jamcells, while FIG. 18B shows the cytotoxcity of the saps on the Jam cellsin the presence of the PKC inhibitor, bisindolymaleimide, and FIG. 18Creveals the cytotoxic effects of saps derived from two Euphorbiaceae onthe Jam cells in the presence of phorbol dibutyrate.

FIG. 19 is a diagrammatic representation of a system used to carry outthe instructions encoded by the storage medium of FIGS. 9 and 10.

FIG. 20 is a diagrammatic representation of a cross-section of amagnetic storage medium.

FIG. 21 is a diagrammatic representation of a cross-section of anoptically readable data storage system.

Compounds may be referred to in the subject specification by a compoundcode. These are defined as below:

TABLE OF COMPOUND CODES COMPOUND CODE DESCRIPTION PEP001 Crude sapPEP002 Methanol and ether extract of E. peplus sap prepared according toExample 7 of PCT/AU98/00656 PEP003 Ingenane enriched fraction preparedaccording to Examples 21 and 23 PEP004 Jatrophane/Pepluane enrichedfraction prepared according to Example 7 of PCT/AU98/00656 PEP005Ingenol-3-angelate PEP006 20-deoxy-ingenol-3-angelate PEP00820-O-acetyl-ingenol-3-angelate PEP009 Acetone Extract of XAD preparedaccording to Example 21 PEP010 Ingenane enriched fraction preparedaccording to Examples 22 and 23

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated in part on the identification ofbiologically useful properties of chemical agents and chemical fractionscomprising these agents obtainable from a member of the Euphorbiaceaefamily of plants or their botanical or horticultural relatives. Thesebiologically useful properties include their use in the prophylaxisand/or treatment of infection, colonization or presence of a particularpathological entity or potential pathological entity or an entitycapable of opportunistic infection or colonization in a particularsubject as well as the amelioration of symptoms associated with orproduced from such an entity.

The term “treatment” is used in its broadest sense and includes theprevention of infection or growth of an entity to pathological levels,the inhibition in growth or reduction in levels of an entity frompathological levels to asymptomatic levels or to levels in between aswell as facilitating the amelioration of the effects of symptoms ofinfection, colonization or presence of an entity.

The term “prophylaxis” is also used herein in its broadest sense toencompass a reduction in the risk of development of infection,colonization or presence of an entity to pathological levels. In certainconditions, an agent may act to treat a subject prophylactically.Furthermore, the prophylactic administration of an agent may result inthe agent becoming involved in the treatment of a pathologicalcondition. Use of the terms “treatment” or “prophylaxis” is not to betaken as limiting the intended result which is to reduce the incidenceof infection, colonization or presence of a pathological entity, apotentially pathogenic entity or an entity capable of opportunisticinfection, colonization or presence or to reduce the effects and/or toameliorate the symptoms or risk of development of symptoms caused orfacilitated by the infection, colonization or presence of the entity.

Furthermore, the symptoms of a pathological condition induced orfacilitated by the infection, colonization or presence of a particularentity may be due to the entity itself or to a toxin,membrane-associated moiety, a soluble or releasable moiety, venom orother molecule associated with the entity. The treatment and prophylaxisof the condition extends, therefore, to the treatment or prophylaxis ofsuch extraneous factors which may persist after the entity has beenremoved or lowered to non-pathological-causing levels.

The present invention is particularly directed to the use of one or moremacrocyclic diterpenes from a member of the Euphorbiaceae family ofplants or botanical or horticultural relatives of such plants. Referenceherein to a member of the Euphorbiaceae family includes reference tospecies from the genera Acalypha, Acidoton, Actinostemon, Adelia,Adenocline, Adenocrepis, Adenophaedra, Adisca, Agrostistachys,Alchornea, Alchorneopsis, Alcinaeanthus, Alcoceria, Aleurites, Amanoa,Andrachne, Angostyles, Anisophyllum, Antidesma, Aphora, Aporosa,Aporosella, Argythamnia, Astrococcus, Astrogyne, Baccanrea,Baliospermum, Bernardia, Beyeriopsis, Bischofia, Blachia, Blumeodondron,Bonania, Bradleia, Breynia, Breyniopsis, Briedelia, Buraeavia,Caperonia, Caryodendron, Celianella, Cephalocroton, Chaenotheca,Chaetocarpus, Chamaesyce, Cheilosa, Chiropetalum, Choriophyllum, Cicca,Chaoxylon, Cleidon, Cleistanthus, Cluytia, Cnesmone, Cnidoscolus,Coccoceras, Codiaeum, Coelodiscus, Conami, Conceveiba, Conceveibastrum,Conceveïbum, Corythea, Croizatia, Croton, Crotonopsis, Crozophora,Cubanthus, Cunuria, Dactylostemon, Dalechampia, Dendrocousinsia,Diaspersus, Didymocistus, Dimorphocalyx, Discocarpus, Ditaxis,Dodecastingma, Drypetes, Dysopsis, Elateriospermum, Endadenium,Endospermum, Erismanthus, Erythrocarpus, Erythrochilus, Eumecanthus,Euphorbia, Euphorbiodendron, Excoecaria, Flueggea, Calearia, Garcia,Gavarretia, Gelonium, Giara, Givotia, Glochidion, Clochidionopsis,Glycydendron, Gymnanthes, Gymnosparia, Haematospermum, Hendecandra,Hevea, Hieronima, Hieronyma, Hippocrepandra, Homalanthus, Hymenocardia,Janipha, Jatropha, Julocroton, Lasiocroton, Leiocarpus, Leonardia,Lepidanthus, Leucocroton, Mabea, Macaranga, Mallotus, Manihot, Mappa,Maprounea, Melanthesa, Mercurialis, Mettenia, Micrandra, Microdesmis,Microelus, Microstachy, Maocroton, Monadenium, Mozinna, Neoscortechinia,Omalanthus, Omphalea, Ophellantha, Orbicularia, Ostodes, Oxydectes,Palenga, Pantadenia, Paradrypeptes, Pausandra, Pedilanthus, Pera,Peridium, Petalostigma, Phyllanthus, Picrodendro, Pierardia,Pilinophytum, Pimeleodendron, Piranhea, Platygyna, Plukenetia,Podocalyx, Poinsettia, Poraresia, Prosartema, Pseudanthus, Pycnocoma,Quadrasia, Reverchonia, Richeria, Richeriella, Ricinella, Ricinocarpus,Rottlera, Sagotia, Sanwithia, Sapium, Savia, Sclerocroton, Sebastiana,Securinega, Senefeldera, Senefilderopsis, Serophyton, Siphonia,Spathiostemon, Spixia, Stillingia, Strophioblachia, Synadenium,Tetracoccus, Tetraplandra, Tetrorchidium, Thyrsanthera, Tithymalus,Trageia, Trewia, Trigonostemon, Tyria and Xylophylla.

The most preferred genus and most suitable for the practice of thepresent invention is the genus Euphorbia. Particularly useful species ofthis genus include Euphorbia aaron-rossii, Euphorbia abbreviata,Euphorbia acuta, Euphorbia alatocaulis, Euphorbia albicaulis, Euphorbiaalgomarginata, Euphorbia aliceae, Euphorbia alta, Euphorbiaanacampseros, Euphorbia andromedae, Euphorbia angusta, Euphorbiaanthonyi, Euphorbia antiguensis, Euphorbia apocynifolia, Euphorbiaarabica, Euphorbia ariensis, Euphorbia arizonica, Euphorbia arkansana,Euphorbia arteagae, Euphorbia arundelana, Euphorbia astroites, Euphorbiaatrococca, Euphorbia baselicis, Euphorbia batabanensis, Euphorbiabergeri, Euphorbia bermudiana, Euphorbia bicolor, Euphorbia biformis,Euphorbia bifurcata, Euphorbia bilobata, Euphorbia biramensis, Euphorbiabiuncialis, Euphorbia blepharostipula, Euphorbia blodgetti, Euphorbiaboerhaavioides, Euphorbia boliviana, Euphorbia bracei, Euphorbiabrachiata, Euphorbia brachycera, Euphorbia brandegee, Euphorbiabrittonii, Euphorbia caesia, Euphorbia calcicola, Euphorbia campestris,Euphorbia candelabrum, Euphorbia capitellata, Euphorbia carmenensis,Euphorbia carunculata, Euphorbia cayensis, Euphorbia celastroides,Euphorbia chalicophila, Euphorbia chamaerrhodos, Euphorbia chamaesula,Euphorbia chiapensis, Euphorbia chiogenoides, Euphorbia cinerascens,Euphorbia clarionensis, Euphorbia colimae, Euphorbia colorata, Euphorbiacommutata, Euphorbia consoquitlae, Euphorbia convolvuloides, Euphorbiacorallifera, Euphorbia creberrima, Euphorbia crenulata, Euphorbiacubensis, Euphorbia cuspidata, Euphorbia cymbiformis, Euphorbiadarlingtonii, Euphorbia defoliata, Euphorbia degeneri, Euphorbiadeltoidea, Euphorbia dentata, Euphorbia depressa Euphorbia dictyosperma,Euphorbia dictyosperma, Euphorbia dioeca, Euphorbia discoidalis,Euphorbia dorsiventralis, Euphorbia drumondii, Euphorbia duclouxii,Euphorbia dussii, Euphorbia eanophylla, Euphorbia eggersii, Euphorbiaeglandulosa, Euphorbia elata, Euphorbia Enola, Euphorbia eriogonoides,Euphorbia eriophylla, Euphorbia esculaeformis, Euphorbia espirituensis,Euphorbia esula, Euphorbia excisa, Euphorbia exclusa, Euphorbiaexstipitata, Euphorbia exstipulata, Euphorbia fendleri, Euphorbiafilicaulis, Euphorbia filiformis, Euphorbia florida, Euphorbiafruticulosa, Euphorbia garber, Euphorbia gaumerii, Euphorbia gerardiana,Euphorbia geyeri, Euphorbia glyptosperma, Euphorbia gorgonis, Euphorbiagracilior, Euphorbia gracillima, Euphorbia gradyi, Euphorbia graminea,Euphorbia graminiea Euphorbia grisea, Euphorbia guadalajarana, Euphorbiaguanarensis, Euphorbia gymnadenia, Euphorbia haematantha, Euphorbiahedyotoides, Euphorbia heldrichii, Euphorbia helenae, Euphorbia helleri,Euphorbia helwigii, Euphorbia henricksonii, Euphorbia heterophylla,Euphorbia hexagona, Euphorbia hexagonoides, Euphorbia hinkleyorum,Euphorbia hintonii, Euphorbia hirtula, Euphorbia hirta, Euphorbiahooveri, Euphorbia humistrata, Euphorbia hypericifolia, Euphorbiainundata, Euphorbia involuta, Euphorbia jaliscensis, Euphorbia jejuna,Euphorbia johnston, Euphorbia juttae, Euphorbia knuthii, Euphorbialasiocarpa, Euphorbia lata, Euphorbia latazi, Euphorbia latericolor,Euphorbia laxiflora Euphorbia lecheoides, Euiphorbia ledienii, Euphorbialeucophylla, Euphorbia lineata, Euphorbia linguiformis, Euphorbialongecornuta, Euphorbia longepetiolata, Euphorbia longeramosa, Euphorbialonginsulicola, Euphorbia longipila, Euphorbia lupulina, Euphorbialurida, Euphorbia lycioides, Euphorbia macropodoides, macvaughiana,Euphorbia manca, Euphorbia mandoniana, Euphorbia mangleti, Euphorbiamango, Euphorbia marylandica, Euphorbia mayana, Euphorbia melanadenia,Euphorbia melanocarpa, Euphorbia meridensis, Euphorbia mertonii,Euphorbia mexiae, Euphorbia microcephala, Euphorbia microclada,Euphorbia micromera, Euphorbia misella, Euphorbia missurica, Euphorbiamontana, Euphorbia montereyana, Euphorbia multicaulis, Euphorbiamultiformis, Euphorbia multinodis, Euphorbia multiseta, Euphorbiamuscicola, Euphorbia neomexicana, Euphorbia nephradenia, Euphorbianiqueroana, Euphorbia oaxacana, Euphorbia occidentalis, Euphorbiaodontodenia, Euphorbia olivacea, Euphorbia olowaluana, Euphorbiaopthalmica, Euphorbia ovata, Euphorbia pachypoda, Euphorbia pachyrhiza,Euphorbia padifolia, Euphorbia palmeri, Euphorbia paludicola, Euphorbiaparciflora, Euphorbia parishii, Euphorbia parryi, Euphorbia paxiana,Euphorbia pediculifera, Euphorbia peplidion, Euphorbia peploides,Euphorbia peplus, Euphorbia pergamena, Euphorbia perlignea, Euphorbiapetaloidea, Euphorbia petaloidea, Euphorbia petrina, Euphorbiapicachensis, Euphorbia pilosula, Euphorbia pilulifera, Euphorbiapinariona, Euphorbia pinetorum, Euphorbia pionosperma, Euphorbiaplatysperma, Euphorbia plicata, Euphorbia poeppigii, Euphorbiapoliosperma, Euphorbia polycarpa, Euphorbia polycnemoides, Euphorbiapolyphylla, Euphorbia portoricensis, Euphorbia portulacoides Euphorbiaportulana, Euphorbia preslii, Euphorbia prostrata, Eulphorbiapteroneura, Euphorbia pycnanthema, Euphorbia ramosa, Euphorbia rapulum,Euphorbia remyi, Euphorbia retroscabra, Euphorbia revoluta, Euphorbiarivularis, Euphorbia robusta, Euphorbia romosa, Euphorbia rubida,Euphorbia rubrosperma, Euphorbia rupicola, Euphorbia sanmartensis,Euphorbia saxatilis M. Bieb, Euphorbia schizoloba, Euphorbiasclerocyathium, Euphorbia scopulorum, Euphorbia senilis, Euphorbiaserpyllifolia, Euphorbia serrula, Euphorbia setiloba Engelm, Euphorbiasonorae, Euphorbia soobyi, Euphorbia sparsiflora, Euphorbiasphaerosperma, Euphorbia syphilitica, Euphorbia spruceana, Euphorbiasubcoerulea, Euphorbia stellata, Euphorbia submammilaris, Euphorbiasubpeltata, Euphorbia subpubens, Euphorbia subreniforme, Euphorbiasubtrifoliata, Euphorbia succedanea, Euphorbia tamaulipasana, Euphorbiatelephioides, Euphorbia tenuissima, Euphorbia tetrapora, Euphorbiatirucalli, Euphorbia tomentella, Euphorbia tomentosa, Euphorbiatorralbasii, Euphorbia tovariensis, Euphorbia trachysperma, Euphorbiatricolor, Euphorbia troyana, Euphorbia tuerckheimii, Euphorbiaturczaminowii, Euphorbia umbellulata, Euphorbia undulata, Euphorbiavermiformis, Euphorbia versicolor, Euphorbia villifera, Euphorbiaviolacea, Euphorbia whitei, Euphorbia xanti Engelm, Euphorbia xylopodaGreemn., Euphorbia yayalesia Urb., Euphorbia yungasensis, Euphorbiazeravschanica and Euphorbia zinniiflora.

Particularly preferred species of the genus Synadenium includeSynadenium grantii and Synadenium compactum.

Particularly preferred species of the genus Monadenium includeMonadenium lugardae and Monadenium guentheri.

A preferred species of the genus Endadenium is Endadenium gossweileni.

Euphorbia peplus is particularly useful in the practice of the presentinvention. Reference herein to “Euphorbia peplus” or its abbreviation“E. peplus” includes various varieties, strains, lines, hybrids orderivatives of this plant as well as its botanical or horticulturalrelatives. Furthermore, the present invention may be practiced using awhole Euphorbiaceae plant or parts thereof including sap or seeds orother reproductive material may be used. Generally, for seeds orreproductive material to be used, a plant or plantlet is first requiredto be propagated.

Reference herein to a Euphorbiaceae plant, a Euphorbia species or E.peplus further encompasses genetically modified plants. Geneticallymodified plants include trangenic plants or plants in which a trait hasbeen removed or where an endogenous gene sequence has beendown-regulated, mutated or otherwise altered including the alteration orintroduction of genetic material which exhibits a regulatory effect on aparticular gene. Consequently, a plant which exhibits a character notnaturally present in a Euphorbiaceae plant or a species of Euphorbia orin E. peplus is nevertheless encompassed by the present invention and isincluded within the scope of the above-mentioned terms.

The macrocyclic diterpenes are generally in extracts of theEuphorbiaceae plants. An extract may comprise, therefore, sap or liquidor semi-liquid material exuded from, or present in, leaves, stem,flowers, seeds, bark or between the bark and the stem. Most preferably,the extract is from sap. Furthermore, the extract may comprise liquid orsemi-liquid material located in fractions extracted from sap, leaves,stems, flowers, bark or other plant material of the Euphoriaceae plant.For example, plant material may be subject to physical manipulation todisrupt plant fibres and extracellular matrix material and inter- andintra-tissue extracted into a solvent including an aqueous environment.All such sources of the macrocyclic diterpenes are encompassed by thepresent invention including macrocyclic diterpenes obtained by syntheticroutes.

The preferred macrocyclic diterpenes are selected from compounds of theingenane, pepluane and jatrophane families. A compound is stated to be amember of the ingenane, pepulane or jatrophane families on the basis ofchemical structure and/or chemical or physical properties. A compoundwhich is a derivative of an ingenane, pepluane or jatrophane isnevertheless encompassed by the present invention through use of theterms “ingenane”, “pepluane” or “jatrophane” since these terms includederivatives, chemical analogues and chemically synthetic forms of thesefamilies of compounds. One particularly preferred derivative is anangeloyl derivative of ingenane.

The preferred chemical agent of the present invention is one whichexhibits an effect on a protein kinase C (PKC) enzyme. Such an effectmay be a direct activation or inhibition of PKC activity or a directeffect on the levels of PKC enzyme in a cell or exported from a cell.Furthermore, the effect may be transitory or may involve an initialactivation of PKC activity or PKC enzyme synthesis or induction of afunctional conformation followed by a down-regulation of PKC activity,enzyme levels or formation of a deactivated conformation. Consequently,an effect on PKC is regarded herein as a modulatory effect and isconveniently determined by consequential events such as resulting fromaltered signal transduction. For example, activation of latent virus,activation of immune mechanisms or activation of a gene promoter mayoccur and this is regarded herein as a modulatory effect on PKC.

The chemical agents of the present invention may be in purified orisolated form meaning that the preparation is substantially devoid ofother compounds or contaminating agents other than diluent, solvent orcarrier or isoforms of the agents. Furthermore, the term “chemicalagent” includes preparations of two or more compounds either admixedtogether or co-purified from a particular source. The chemical agent mayalso be a chemical fraction, extract or other preparation from theEuphorbiaceace plant.

Consequently, reference herein to a “chemical agent” includes a purifiedform of one or more compounds or a chemical fraction or extract such asfrom the sap of a Euphorbiaceace plant, and in particular a species ofEuphorbia, and most preferably from E. peplus or botanical orhorticultural relatives or variants thereof.

Accordingly, one aspect of the present invention contemplates a methodfor the treatment or prophylaxis of a condition associated with thepresence of a biological entity or part thereof or a toxin or venomtherefrom or a genetic event caused thereby in a subject, said methodcomprising the administration to said subject of a symptom-amelioratingeffective amount of a chemical agent obtainable from a plant of theEuphorbiaceae family or a derivative or chemical analogue thereof whichchemical agent is a macrocyclic diterpene selected from compounds of theingenane, pepluane and jatrophane families and which chemical agent orderivative or chemical analogue is represented by any one of the generalformulae (I)-(V)

wherein:

-   -   n is 0-10 atoms selected from carbon, oxygen, nitrogen, sulfur,        phosphorus, silicon, boron, arsenic and selenium, wherein the        ring defined by said atoms is saturated or unsaturated,        including epoxides and thioepoxides;    -   A-T are independently selected from hydrogen, R₁, R₂, R₃, F, Cl,        Br, I, CN, OR₁, SR₁, NR₁R₂, N(═O)₂, NR₁OR₂, ONR₁R₂, SOR₁, SO₂R₁,        SO₃R₁, SONR₁R₂, SO₂NR₁R₂, SO₃NR₁R₂, P(R₁)₃, P(═O)(R₁)₃, Si(R₁)₃,        B(R₁)₂, (C═X)R₃ or X(C═X)R₃ where X is selected from sulfur,        oxygen and nitrogen;    -   R₁ and R₂ are each independently selected from C₁-C₂₀ alkyl        (branched and/or straight chained), C₁-C₂₀ arylalkyl, C₃-C₈        cycloalkyl, C₆-C₁₄ aryl, C₁-C₁₄ heteroaryl, C₁-C₁₄ heterocycle,        C₂-C₁₀ alkenyl (branched and/or straight chained), C₂-C₁₀        alkynyl (branched and/or straight chained), C₁-C₁₀        heteroarylalkyl, C₁-C₁₀ alkoxyalkyl, C₁-C₁₀ haloalkyl,        dihaloalkyl, trihaloalkyl, haloalkoxy, C₁-C₁₀ [CN, OR₁, SR₁,        NR₁R₂, N(═O)₂, NR₁OR₂, ONR₁R₂, SOR₁, SO₂R₁, SO₃R₁, SONR₁R₂,        SO₂NR₁R₂, SO₃NR₁R₂, P(R₁)₃, P(═O)(R₁)₃, Si(R₁)₃, B(R₁)₂]alkyl;    -   R₃ is selected from R₁, R₂, CN, COR₁, CO₂R₁, OR₁, SR₁, NR₁R₂,        N(═O)₂, NR₁OR₂, ONR₁R₂, SOR₁, SO₂R₁, SO₃R₁, SONR₁R₂, SO₂NR₁R₂,        SO₃NR₁R₂, P(R₁)₃, P(═O)(R₁)₃, Si(R₁)₃, B(R₁)₂;    -   A connected to B (or C), D (or E), R (or Q), P (or O) or S        (or T) is a selection of C₁-C₈ disubstituted (fused) saturated        or unsaturated carbocyclic or heterocyclic rings further        substituted by R₃, (C═X)R₃ and X(C═X)R₃, including epoxides and        thioepoxides;    -   J connected to I (or H), G (or F), K (or L), M (or N) or S        (or T) is a selection of C₁-C₈ disubstituted (fused) saturated        and unsaturated carbocyclic or heterocyclic rings further        substituted by R₃, (C═X)R₃ and X(C═X)R₃, including epoxides and        thioepoxides;    -   D (or E) connected to B (or C) or G (or F); I (or H) connected        to G (or F); P (or O) connected to R (or Q) or M (or N); K        (or L) connected to N (or M) is a selection of C₁-C₈        disubstituted (fused) saturated or unsaturated carbocyclic or        heterocyclic rings substituted by R₃, (C═X)R₃ and X(C═X)R₃,        including epoxides and thioepoxides;    -   B and C, D and E, R and Q, P and O, I and H, G and F, K and L, M        and N or S and T are ═X where X is selected from sulfur, oxygen,        nitrogen, NR₁R₂, and ═CR₁R₂

wherein:

-   -   n is 0-10 atoms selected from carbon, oxygen, nitrogen, sulfur,        phosphorus, silicon, boron, arsenic and selenium, wherein the        ring defined by said atoms is saturated or unsaturated,        including epoxides and thioepoxides;    -   A′-T′ are independently selected from hydrogen, R₄, R₅, R₆, F,        Cl, Br, I, CN, COR₄, CO₂R₄, OR₄, SR₄, NR₄R₅, CONR₄R₅, N(═O)₂,        NR₄OR₅, ONR₄R₅, SOR₄, SO₂R₄, SO₃R₄, SONR₄R₅, SO₂NR₄R₅, SO₃NR₄R₅,        P(R₄)₃, P(═O)(R₄)₃, Si(R₄)₃, B(R₄)₂, (C═X)R₆ or X(C═X)R₆ where X        is selected from sulfur, oxygen and nitrogen;    -   R₄ and R₅ are each independently selected from C₁-C₂₀ alkyl        (branched and/or straight chained), C₁-C₂₀ arylalkyl, C₃-C₈        cycloalkyl, C₆-C₁₄ aryl, C₁-C₁₄ heteroaryl, C₁-C₁₄ heterocycle,        C₂-C₁₀ alkenyl (branched and/or straight chained), C₂-C₁₀        alkynyl (branched and/or straight chained), C₁-C₁₀        heteroarylalkyl, C₁-C₁₀ alkoxyalkyl, C₁-C₁₀ haloalkyl,        dihaloalkyl, trihaloalkyl, haloalkoxy, C₁-C₁₀ [CN, OR₄, SR₄,        NR₄R₅, N(═O)₂, NR₄OR₅, ONR₄R₅, SOR₄, SO₂R₄, SO₃R₄, SONR₄R₅,        SO₂NR₄R₅, SO₃NR₄R₅, P(R₄)₃, P(═O)(R₄)₃, Si(R₄)₃, B(R₄)₂]alkyl;    -   R₆ is selected from R₄, R₅, CN, COR₄, CO₂R₄, OR₄, SR₄, NR₄R₅,        N(═O)₂, NR₄OR₅, ONR₄R₅, SOR₄, SO₂R₄, SO₃R₄, SONR₄R₅, SO₂NR₄R₅,        SO₃NR₄R₅, P(R₄)₃, P(═O)(R₄)₃, Si(R₄)₃, B(R₄)₂;    -   E′ and R′ or H′ and O′ is a C₂-C₈ saturated or unsaturated        carbocyclic or heterocyclic ring system further substituted by        R₆, including epoxides and thioepoxides;    -   O′ connected to M′ (or N′) or Q′ (or P′); R′ connected to Q′ (or        P′) or S′ (or T′); S′ (or T′) connected to A′ (or B′); A′ (or        B′) connected to C′ (or D′); E′ connected to C′ (or D′) or F′        (or G′); H′ connected to I′; I′ connected to J′; J′ connected to        K′; K′ connected to L′; L′ connected to M′ (or N′) are C₁-C₈        disubstituted (fused) saturated or unsaturated carbocyclic or        heterocyclic ring systems further substituted by R₆, (C═X)R₆ and        X(C═X)R₆, including epoxides and thioepoxides;    -   A′, B′ and C′, D′ and F′, G′ and M′, N′ and P′, Q′ and S′, T′        are ═X where X is selected from sulfur, oxygen, nitrogen, NR₄R₅,        (C═X)R₆, X(C═X)R₆, and ═CR₇R₈;        R₇ and R₈ are each independently selected from R₆, (C═X)R₆ and        X(C═X)R₆

wherein:

-   -   n is 0-10 atoms selected from carbon, oxygen, nitrogen, sulfur,        phosphorus, silicon, boron, arsenic and selenium, wherein the        ring defined by said atoms is saturated or unsaturated,        including epoxides and thioepoxides;    -   A¹-T¹ are independently selected from hydrogen, R₉, R₁₀, R₁₁, F,        Cl, Br, I, CN, OR₉, SR₉, NR₉R₁₀, N(═O)₂, NR₉OR₁₀, ONR₉R₁₀, SOR₉,        SO₂R₉, SO₃R₉, SONR₉R₁₀, SO₂NR₉R₁₀, SO₃NR₉R₁₀, P(R₉)₃,        P(═O)(R₉)₃, Si(R₉)₃, B(R₉)₂, (C═X)R₁₁ or X(C═X)R₁₁ where X is        selected from sulfur, oxygen and nitrogen;    -   R₉ and R₁₀ are each independently selected from C₁-C₂₀ alkyl        (branched and straight chained), C₁-C₂₀ arylalkyl, C₃-C₈        cycloalkyl, C₆-C₁₄ aryl, C₁-C₁₄ heteroaryl, C₁-C₁₄ heterocycle,        C₂-C₁₀ alkenyl (branched and straight chained), C₂-C₁₀ alkynyl        (branched and straight chained), C₁-C₁₀ heteroarylalkyl, C₁-C₁₀        alkoxyalkyl, C₁-C₁₀ haloalkyl, dihaloalkyl, trihaloalkyl,        haloalkoxy, C₁-C₁₀ [CN, OR₉, SR₉, NR₉R₁₀, N(═O)₂, NR₉OR₁₀,        ONR₉R₁₀, SOR₉, SO₂R₉, SO₃R₉, SONR₉R₁₀, SO₂NR₉R₁₀, SO₃NR₉R₁₀,        P(R₉)₃, P(═O)(R₉)₃, Si(R₉)₃, B(R₉)₂]alkyl;    -   R₁₁ is selected from R₉, R₁₀, CN, COR₉, CO₂R₉, OR₉, SR₉, NR₉R₁₀,        N(═O)₂, NR₉OR₁₀, ONR₉R₁₀, SOR₉, SO₂R₉, SO₃R₉, SONR₉R₁₀,        SO₂NR₉R₁₀, SO₃NR₉R₁₀, P(R₉)₃, P(═O)(R₉)₃, Si(R₉)₃, B(R₉)₂;    -   B¹ and R₁, E¹ and Ö¹ and Ë¹ and M¹ are selected from a C₂-C₈        saturated or unsaturated carbocyclic or heterocyclic ring system        further substituted by R₁₁, including epoxides and thioepoxides;    -   A¹ (or Ä¹) connected to Á¹ (or Ã¹) or T¹ (or S¹); B¹ connected        to Á¹ (or Ã¹) or C¹ (or D¹). E¹ connected to Ë¹ or C¹ (or D¹);        Ë¹ connected to É¹ (or F¹); G¹ (or H¹) connected to É¹ (or F¹)        or I¹ (or J¹); K¹ (or L¹) connected to I¹ (or J¹) or M¹; M¹        connected to O¹ (or N¹); Ö¹ connected O¹ (or N¹) or P¹ (or Q¹);        R¹ connected P¹ (or Q¹) or S¹ (or T¹) are C₁-C₈ disubstituted        (fused) saturated or unsaturated carbocyclic or heterocyclic        ring systems further substituted by R₁₁, (C═X)R₁₁ and X(C═X)R₁₁,        including epoxides and thioepoxides;    -   A¹, Ä and Á, Ã and C¹, D¹ and F¹, É and G¹, H¹ and I¹, J¹ and        K¹, L¹ and N¹, O¹ and P¹, Q¹ and S¹, T¹ are ═X where X is        selected from sulfur, oxygen, nitrogen, NR₉R₁₀, including        (C═X)R₁₁ and X(C═X)R₁₁, and ═CR₁₂R₁₃;    -   R₁₂ and R₁₃ are independently selected from R₁₁ , (C═X)R₁₁ and        X(C═X)R₁₁

wherein:

-   -   n is 0-10 atoms selected from carbon, oxygen, nitrogen, sulfur,        phosphorus, silicon, boron, arsenic and selenium, wherein the        ring defined by said atoms is saturated or unsaturated,        including epoxides and thioepoxides;    -   A²-X² are independently selected from hydrogen, R₁₄, R₁₅, R₁₆,        F, Cl, Br, I, CN, OR₁₄, SR₁₄, NR₁₄R₁₅, N(═O)₂, NR₁₄OR₁₅,        ONR₁₄R₁₅, SOR₁₄, SO₂R₁₄, SO₃R₁₄, SONR₁₄R₁₅, SO₂NR₁₄R₁₅,        SO₃NR₁₄R₁₅, P(R₁₄)₃, P(═O)(R₁₄)₃, Si(R₁₄)₃, B(R₁₄), (C═Y)R₁₆ or        Y(C═Y)R₁₆ where Y is selected from sulfur, oxygen and nitrogen;    -   R₁₄ and R₁₅ are each independently selected from C₁-C₂₀ alkyl        (branched and/or straight chained), C₁-C₂₀ arylalkyl, C₃-C₈        cycloalkyl, C₆-C₁₄ aryl, C₁-C₁₄ heteroaryl, C₁-C₁₄ heterocycle,        C₂-C₁₀ alkenyl (branched and/or straight chained), C₂-C₁₀        alkynyl (branched and/or straight chained), C₁-C₁₀        heteroarylalkyl, C₁-C₁₀ alkoxyalkyl, C₁-C₁₀ haloalkyl,        dihaloalkyl, trihaloalkyl, haloalkoxy, C₁-C₁₀ [CN, OR₁₄, SR₁₄,        NR₁₄R₁₀, N(═O)₂, NR₁₄OR₁₅, ONR₁₄R₁₅, SOR₁₄, SO₂R₁₄, SO₃R₁₄,        SONR₁₄R₁₅, SO₂NR₁₄R₁₅, SO₃NR₁₄R₁₅, P(R₁₄)₃, P(═O)(R₁₄)₃,        Si(R₁₄)₃, B(R₁₄)₂]alkyl;    -   R₁₆ is selected from R₁₄, R₁₅, CN, COR₁₄, CO₂R₁₅, OR₁₄, SR₁₄,        NR₁₄R₁₅, N(═O)₂, NR₁₄OR₁₅, ONR₁₄R₁₅, SOR₁₄, SO₂R₁₄, SO₃R₁₄,        SONR₁₄R₁₅, SO₂NR₁₄R₁₅, SO₃NR₁₄R₁₅, P(R₁₄)₃, P(═O)(R₁₄)₃,        Si(R₁₄)₃, B(R₁₄)₂;    -   E² and V², H² and S², and I² and P² are C₂-C₈ saturated or        unsaturated carbocyclic or heterocyclic ring system further        substituted by R₁₆, including epoxides and thioepoxides;    -   A² (or B²) connected to C² (or D²) or W² (or X²); E² connected        to C² (or D²) or F² (or G²); H² connected to F² (or G²) or I²;        I² connected to J² (or K²); L² (or M²) connected to J² (or K²)        or N² (or O²); R² (or Q²) connected to P² or S²; V² connected to        U² (or T²) or W² (or X²) are C₁-C₈ disubstituted (fused)        saturated or unsaturated carbocyclic or heterocyclic ring        systems further substituted by R₁₆, (C═Y)R₁₆ and Y(C═Y)R₁₆,        including epoxides and thioepoxides;    -   A², B²; C², D²; F², G²; J², K²; L², M²; N², O²; Q², R²; U², T²        and X², W² are ═Y where Y is selected from sulfur, oxygen,        nitrogen, NR₁₄R₁₅ and ═CR₁₇R₁₈;    -   R₁₇ and R₁₈ are independently selected from R₁₆, (C═Y)R₁₆ and        Y(C═Y)R₁₆

wherein:

-   -   n is 0-10 atoms selected from carbon, oxygen, nitrogen, sulfur,        phosphorus, silicon, boron, arsenic and selenium, wherein the        ring defined by said atoms is saturated or unsaturated,        including epoxides and thioepoxides;    -   A³-Z³ are independently selected from hydrogen, R₁₉, R₂₀, R₂₁,        F, Cl, Br, I, CN, OR₁₉, SR₁₉, NR₁₉R₂₀, N(═O)₂, NR₁₉OR₂₀,        ONR₁₉R₂₀, SOR₁₉, SO₂R₁₉, SO₃R₁₉, SONR₁₉R₂₀, SO₂NR₁₉R₂₀,        SO₃NR₁₉R₂₀, P(R₁₉)₃, P(═O)(R₁₉)₃, Si(R₁₉)₃, B(R₁₉)₂, (C═Ø)R₂₁ or        Ø(C═Ø)R₂₁ where Ø is sulfur, oxygen and nitrogen;    -   R₁₉ and R₂₀ are each independently selected from C₁-C₂₀ alkyl        (branched and/or straight chained), C₁-C₂₀ arylalkyl, C₃-C₈        cycloalkyl, C₆-C₁₄ aryl, C₁-C₁₄ heteroaryl, C₁-C₁₄ heterocycle,        C₂-C₁₀ alkenyl (branched and/or straight chained), C₂-C₁₀        alkynyl (branched and/or straight chained), C₁-C₁₀        heteroarylalkyl, C₁-C₁₀ alkoxyalkyl, C₁-C₁₀ haloalkyl,        dihaloalkyl, trihaloalkyl, haloalkoxy, C₁-C₁₀ [CN, OR₁₉, SR₁₉,        NR₁₉R₂₀, N(═O)₂, NR₁₉OR₂₀, ONR₁₉R₂₀, SOR₁₉, SO₂R₁₉, SO₃R₁₉,        SONR₁₉R₂₀, SO₂NR₁₉R₂₀, SO₃NR₁₉R₂₀, P(R₁₉)₃, P(═O)(R₁₉)₃,        Si(R₁₉)₃, B(R₁₉)₂]alkyl;    -   R₂₁ is selected from R₁₉, R₂₀, CN, COR₁₉, CO₂R₁₉, OR₁₉, SR₁₉,        NR₁₉R₂₀, N(═O)₂, NR₁₉OR₂₀, ONR₁₉R₂₀, SOR₁₉, SO₂R₁₉, SO₃R₁₉,        SONR₁₉R₂₀, SO₂NR₁₉R₂₀, SO₃NR₁₉R₂₀, P(R₁₉)₃, P(═O)(R₁₉)₃,        Si(R₁₉)₃, B(R₁₉)₂;    -   D³ connected to X³ is a C₂-C₈ saturated or unsaturated        carbocyclic or heterocyclic ring system further substituted by        R₂₁, including epoxides and thioepoxides;        A³ (or Ä³) connected to B³ (or C³) or Z³ (or Y³); D³ connected        to B³ (or C³) or E³ (or F³; G³ (or H³) connected to E³ (or F³)        or I³ (or J³); L³ (or K³) connected to I³ (or J³) or M³ (or N³);        O³ (or Ö³) connected to N³ (or M³) or P³ (or Q³). S³ (or R³)        connected to Q³ (or P³) or U³ (or T³). W³ (or V³) connected to        U³ (or T³) or X³; X³ connected to Y³ (or Z³) are C₁-C₈        disubstituted (fused) saturated or unsaturated carbocyclic or        heterocyclic ring systems further substituted by R₂₁, (C═Ø)R₂₁        and Ø (C═Ø)R₂₁, including epoxides and thioepoxides;    -   A³, Ä³; B³, C³; E³, F³; G³, H³; I³, J³; K³, L³; M³, N³; O³, Ö³;        Q³, P³, S³, R³, U³, T³, W³, V³, and Z³, Y³ are ═Ø where Ø is        selected from sulfur, oxygen, nitrogen, NR₁₉R₂₀, and ═CR₂₂R₂₃;        and    -   R₂₂ and R₂₃ are selected from R₂₁, (C═Ø)R₂₁ and Ø(C═Ø)R₂₁;        and which chemical agent or derivative or chemical analogue        thereof is capable of modulating PKC activity, PKC-dependent        gene expression or PKC enzyme turnover and wherein said chemical        agent or its derivatives or chemical analogues is administered        for a time and under conditions sufficient to ameliorate one or        more symptoms associated with said biological entity.

A preferred compound of Formula I has the formula wherein n is 1 so thatthe rings on which S and T are substituted is a 7-membered ring, S isC═O and S and T together are C═O and R and A and the carbon atoms towhich they are attached form a fused saturated or unsaturated carboxylicor heterocyclic ring, and G and I and the carbon atoms to which they areattached form a 3-membered cyclic ring. Under these circumstances, inone embodiment, the definitions of B, C, D, E, F, H, J, K, L, M, N, O,P, Q are as defined hereinabove. In these circumstances, it is preferredthat they are not hyroxyalkyl, such as hydroxymethyl or alkoxy alkyl,wherein alkyl is as defined herein. In another embodiment, neither ofthe aforementioned variables are hydroxyalkyl wherein alkyl is definedhereinabove. In another embodiment, all of the variables B, C, D, G, F,H, J, K, L, O, P and Q are as defined hereinabove except that neither Mor N are hydroxyalkyl such as hydroxymethyl or hydroxyethyl oralkoxyalkyl. In another embodiment M or N are not hydroxyalkyl. In astill further embodiment, M or N are not hydroxymethyl or hydroxyethyl.

Especially preferred chemical agents or derivatives or chemicalanalogues thereof are represented by the general formula (VI):

wherein:

-   -   R₂₄, R₂₅ and R₂₆ are independently selected from hydrogen, R₂₇,        R₂₈, F, Cl, Br, I, CN, OR₂₇, SR₂₇, NR₂₇R₂₈, N(═O)₂, NR₂₇OR₂₈,        ONR₂₇R₂₈, SOR₂₇, SO₂R₂₇, SO₃R₂₇, SONR₂₇R₂₈, SO₂NR₂₇R₂₈,        SO₃NR₂₇R₂₈, P(R₂₇)₃, P(═O)(R₂₇)₃, Si(R₂₇)₃, B(R₂₇)₂, (C═X)R₂₉ or        X(C═X)R₂₉ where X is selected from sulfur, oxygen and nitrogen;    -   R₂₇ and R₂₈ are each independently selected from C₁-C₂₀ alkyl        (branched and/or straight chained), aryl C₁-C₂₀ alkyl, C₃-C₈        cycloalkyl, C₆-C₁₄ aryl, C₁-C₁₄ heteroaryl, C₁-C₁₄ heterocycle,        C₂-C₁₀ alkenyl (branched and/or straight chained), C₂-C₁₀        alkynyl (branched and/or straight chained), C₁-C₁₀        heteroarylalkyl, C₁-C₁₀ alkoxyalkyl, C₁-C₁₀ haloalkyl,        dihaloalkyl, trihaloalkyl, haloalkoxy, C₁-C₁₀ alkyl which is        unsubstituted or substituted by CN, OR₃₀, SR₃₀, NR₃₀R₃₁, N═(O)₂,        NR₃₀ OR₃₁, ONR₃₀ R₃₁, SOR₃₀, SO₂R₃₀, SO₃R₃₀, SONR₃₀R₃₁,        SO₂NR₃₀R₃₁, SO₃NR₃₀R₃₁, P(R₃₀)₃, P═(O)(R₃₀)₃, Si(R₃₀)₃, or        B(R₃₀)₂;    -   R₂₉ is selected from R₂₇, R₂₈, CN, COR₂₇, CO₂R₂₇, OR₂₇, SR₂₇,        NR₂₇R₂₈, N(═O)₂, NR₂₇OR₂₈, ONR₂₇R₂₈, SOR₂₇, SO₂R₂₇, SO₃R₂₇,        SONR₂₇R₂₈, SO₂NR₂₇R₂₈, SO₃NR₂₇R₂₈, P(R₂₇)₃, P(═O)(R₂₇)₃,        Si(R₂₇)₃, and B(R₂₇)₂;    -   each R₃₀ and R₃₁ are independently C₁-C₂₀ alkyl (branched or        straight chained), aryl C₁-C₂₀ alkyl, C₃-C₈ cycloalkyl, C₆-C₁₄        aryl, C₁-C₁₄-heteroaryl, C₁-C₁₄-heterocycle, C₂-C₁₀ alkenyl        (branched or straight chained), C₂-C₁₀ alkynyl (branched or        straight chained), C₁-C₁₀ heteroarylalkyl, C₁-C₁₀ alkoxyalkyl,        C₁-C₁₀ haloalkyl, dihaloalkyl, trihaloalkyl, or haloalkoxy.

In a preferred embodiment, R₂₄ is hydrogen, OAcetyl or OH.

In another preferred embodiment, R₂₅ and R₂₆ are OH.

In an embodiment, R₂₅ and R₂₆ are not hydroxyalkyl, such ashydroxymethyl or alkoxy alkyl and R₂₄ is not hydroxy, alkoxy,hydroxyalkyl or alkoxy alkyl. In another embodiment R₂₅ and R₂₆ are asdefined hereinabove except they are not hydroxyalkyl and R₂₄ is asdefined hereinabove, except it is not hydroxy, alkoxy, hydroxyalkyl oralkoxy alkyl. In a still further embodiment, R₂₅ and R₂₆ are as definedhereinabove, and R₂₄ is not hydroxy, alkoxy or hydroxyalkyl oralkoxyalkyl. In a still further embodiment, R₂₅ and R₂₆ are as definedhereinabove and R₂₄ is not hydroxy or CH₂OH.

As used herein, the term “alkyl” refers to linear or branched chains.The term “haloalkyl” refers to an alkyl group substituted by at leastone halogen. Similarly, the term “haloalkoxy” refers to an alkoxy groupsubstituted by at least one halogen. As used herein the term “halogen”refers to fluorine, chlorine, bromine and iodine.

As used herein the term “aryl” refers to aromatic carbocyclic ringsystems such as phenyl or naphthyl, anthracenyl, especially phenyl.Suitably, aryl is C₆-C₁₄ with mono, di- and tri-substitution containingF, Cl, Br, I, NO₂, CF₃, CN, OR₁, COR₁, CO₂R₁, NHR₁, NR₁R₂, NR₁OR₂,ONR₁R₂, SOR₁, SO₂R₁, SO₃R₁, SONR₁R₂, SO₂NR₁R₂, SO₃NR₁R₂, P(R₁)₃,P(═O)(R₁)₃, Si(R₁)₃, B(R₁)₂, wherein R₁ and R₂ are defined above

As used herein the terms “heterocycle”, “heterocyclic”, “heterocyclicsystems” and the like refer to a saturated, unsaturated, or aromaticcarbocyclic group having a single ring, multiple fused rings (forexample, bicyclic, tricyclic, or other similar bridged ring systems orsubstituents), or multiple condensed rings, and having at least oneheteroatom such as nitrogen, oxygen, or sulfur within at least one ofthe rings. This term also includes “heteroaryl” which refers to aheterocycle in which at least one ring is aromatic. Any heterocyclic orheteroaryl group can be unsubstituted or optionally substituted with oneor more groups, as defined above. Further, bi- or tricyclic heteroarylmoieties may comprise at least one ring, which is either completely, orpartially, saturated. Suitable heteroaryl moieties include, but are notlimited to oxazolyl, thiazaoyl, thienyl, furyl, 1-isobenzofuranyl,3H-pyrrolyl, 2H-pyrrolyl, N-pyrrolyl, imidazolyl, pyrazolyl,isothiazolyl, isooxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyradazinyl,indolizinyl, isoindolyl, indoyl, indolyl, purinyl, phthalazinyl,1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazoyl, 1,2,4-oxadiazolyl,1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,3,4-oxatriazolyl,1,2,3,5-oxatriazolyl, 1,3,5-triazinyl, 1,2,4-triazinyl, 1,2,3-triazinyl,azepinyl, oxepinyl, thiepinyl, benzofuranyl, isobenzofuranyl,thionaphthenyl, isothionaphthenyl, indoleninyl, 2-isobenzazolyl,1,5-pyrindinyl, pyrano[3,4-b]pyrrolyl, isoindazolyl, indoxazinyl,benzoxazolyl, anthranilyl, quinolinyl, isoquinolinyl, cinnolinyl,quinazolinyl, naphthyridinyl, pyrido[3,4-b]pyridinyl, andpyrido[3,2-b]pyridinyl, pyrido[4,3-b]pyridinyl.

Reference to a biological entity includes a prokaryotic microorganism, alower eukaryotic microorganism, a complex eukaryotic organism or avirus.

A prokaryotic microorganism includes bacteria such as Gram positive,Gram negative and Gram variable bacteria and intracellular bacteria.Examples of bacteria contemplated herein include the speices of thegenera Treponema sp., Borrelia sp., Neisseria sp., Legionella sp.,Bordetella sp., Escherichia sp., Salmonella sp., Shigella sp.,Klebsiella sp., Yersinia sp., Vibrio sp., Hemophilus sp., Rickettsiasp., Chlamydia sp., Mycoplasma sp., Staphylococcus sp., Streptococcussp., Bacillus sp., Clostridium sp., Corynebacterium sp.,Proprionibacterium sp., Mycobacterium sp., Ureaplasma sp. and Listeriasp.

Particularly preferred species include Treponema pallidum, Borreliaburgdorferi, Neisseria gonorrhea, Neisseria meningitidis, Legionellapneumophila, Bordetella pertussis, Escherichia coli, Salmonella typhi,Salmonella typhimurium, Shigella dysenteriae, Klebsiella pneumoniae,Yersinia pestis, Vibrio cholerae, Hemophilus influenzae, Rickettsiarickettsii, Chlamydia trachomatis, Mycoplasma pneumoniae, Staphylococcusaureus, Streptococcus pneumoniae, Streptococcus pyogenes, Bacillusanthracis, Clostridium botulinum, Clostridium tetani, Clostridiumperfringens, Corynebacterium diphtheriae, Proprionibacterium acnes,Mycobacterium tuberculosis, Mycobacterium leprae and Listeriamonocytogenes.

A lower eukaryotic organism includes a yeast or fungus such as but notlimited to Pneumocystis carinii, Candida albicans, Aspergillus,Histoplasma capsulatum, Blastomyces dermatitidis, Cryptococcusneoformans, Trichophyton and Microsporum.

A complex eukaryotic organism includes worms, insects, arachnids,nematodes, aemobe, Entamoeba histolytica, Giardia lamblia, Trichomonasvaginalis, Trypanosoma brucei gambiense, Trypanosoma cruzi, Balantidiumcoli, Toxoplasma gondii, Cryptosporidium or Leishmania.

The term “viruses” is used in its broadest sense to include viruses ofthe families adenoviruses, papovaviruses, herpesviruses: simplex,varicella-zoster, Epstein-Barr, CMV, pox viruses: smallpox, vaccinia,hepatitis B, rhinoviruses, hepatitis A, poliovirus, rubellavirus,hepatitis C, arboviruses, rabiesvirus, influenzaviruses A and B,measlesvirus, mumpsvirus, HIV, HTLV I and II.

Particularly preferred prokaryotic microorganisms are Salmonella sp. andother enteric microorganisms and Streptococcus sp. and Staphylococcussp. Particularly preferred lower eukaryotic organisms include species ofTrichophytos, Microsporum and Epidermophytos, yeast and Plasmodium sp.such as malaria agents.

Preferred complex eukaroytic organisms are insects such as blood-suckinginsects.

Preferred viruses are HIV, EBV and CMV.

As stated above, the present invention extends to the use of the subjectchemical agents to not only directly remove, destroy or reduce thelevels of the entity but also of any molecule associated therewith suchas toxins and venoms. The agents may act directly on the agent orindirectly via, for example, activation of the immune system and/oractivation of latent viruses.

In one particular embodiment, the condition to be treated is associatedwith a prokaryotic microorganism.

Accordingly, another aspect of the present invention contemplates amethod for the treatment or prophylaxis of a microbial infection in asubject, said method comprising the administration to said subject of asymptom-ameliorating effective amount of a macrocyclic diterpene, or achemical fraction comprising same from a plant of the familyEuphorbiaceae or a derivative or chemical analogue of said macrocyclicditerpene having the structures as defined above wherein saidmacrocyclic diterpene or its derivative or chemical analogue modulatesPKC activity, synthesis or enzyme turnover, said administration beingfor a time and under conditions sufficient to ameliorate one or moresymptoms of the infection.

The preferred microorganism in this context includes but is not limitedto Salmonella sp., Streptococcus sp. and Staphylococcus sp.

A “symptom” includes sickness, bacterial loads in particular specimensassociated illness (e.g. headaches, lethargy, rashes, fever),septicemia, bacteremia and inflammation.

In another embodiment, the entity is a lower eukaryotic organism.

Accordingly, another aspect of the present invention provides a methodfor the treatment or prophylaxis of an infection by a lower eukaryoticorganism in a subject, said method comprising the administration to saidsubject of a symptom-ameliorating effective amount of a macrocyclicditerpene or chemical fraction comprising same from a plant of thefamily Euphorbiaceae or a derivative or chemical analogue of saidmacrocyclic diterpene having the structures as defined above whereinsaid macrocyclic diterpene or its derivative or chemical analoguemodulates PKC activity, synthesis or enzyme turnover, saidadministration being for a time and under conditions sufficient toameliorate one or more symptoms of the infection.

Preferably, the lower eukaryotic organism is a yeast or fungi such asbut not limited to Pneumocystis carinii, Candida albicans, Aspergillus,Histoplasma capsulatum, Blastomyces dermatitidis, Cryptococcusneoformans, Trichophyton and Microsporim.

Particularly preferred lower eukaryotic organisms are yeasts, fungi anda Plasmodium sp.

The term “symptom” is as stated above except in relation to eukaryoticorganism.

In yet another embodiment, the entity is a complex eukaryotic organism.

Accordingly, another aspect of the present invention provide a methodfor the treatment or prophylaxis of an infection by a complex eukaryoticorganism in a subject, said method comprising the administration to saidsubject of a symptom-ameliorating effective amount of a macrocyclicditerpene, or a chemical fraction comprising same from a plant of thefamily Euphorbiaceae or a derivative or chemical analogue of saidmacrocyclic diterpene having the structures as defined above whereinsaid macrocyclic diterpene or its derivative or chemical analoguemodulates PKC activity, synthesis or enzyme turnover, saidadministration being for a time and under conditions sufficient toameliorate one or more symptoms of the infection.

Preferably, the complex eukaryotic organism is a worm, insect, arachnid,nematode, aemobe, Entamoeba histolytica, Giardia lamblia, Trichomonasvaginalis, Trypanosoma brucei gambiense, Trypanosoma cruzi, Balantidiumcoli, Plasmodium malariae, Plasmodium tropicalis, Toxoplasma gondii,Cryptosporidium or Leishmania.

In yet another embodiment, the entity is a virus.

Accordingly, another aspect of the present invention provide a methodfor the treatment or prophylaxis of an infection by a virus in asubject, said method comprising the administration to said subject of asymptom-ameliorating effective amount of a macrocyclic diterpene, or achemical fraction comprising same from a plant of the familyEuphorbiaceae or a derivative or chemical analogue of said macrocyclicditerpene having the structures as defined above wherein saidmacrocyclic diterpene or its derivative or chemical analogue modulatesPKC activity, synthesis or enzyme turnover, said administration beingfor a time and under conditions sufficient to ameliorate one or moresymptoms of the infection.

Preferred viruses include adenoviruses, papovaviruses, herpesviruses:simplex, varicella-zoster, Epstein-Barr, CMV, pox viruses: smallpox,vaccinia, hepatitis B, rhinoviruses, hepatitis A, poliovirus,rubellavirus, hepatitis C, arboviruses, rabiesvirus, influenzaviruses Aand B, measlesvirus, mumpsvirus, HIV, HTLV I and II.

This aspect of the present invention is particularly useful in thetreatment of latent virus infection. The term “latent virus” includesreference to a virus or more particularly a virus genome or part thereofwhich has integrated into the genome of a cell. When in the latentstate, it is more difficult for a host's immune system to recognize avirus as a foreign body. In accordance with the present invention, it isproposed that the subject chemical agents are capable of activating alatent virus thereby causing the virus to undergo replication and atleast partial assembly. As a result, a mechanism within the host orwithin the cells of the host is then induced to assist in theeradication of the virus. The present invention extends to both thedirect effect of the chemical agent on the virus as well as promotingthe immune system to direct same against the virus and to combinationtherapies with anti-viral agents.

Accordingly, another aspect of the present invention contemplates amethod for the treatment or prophylaxis of infection by a latent virusin a subject, said method comprising the administration to said subjectof a virus activating effective amount of a macrocyclic diterpene, or achemical fraction comprising same from a plant of the familyEuphorbiaceae or a derivative or chemical analogue of said macrocyclicditerpene having the structures as defined above wherein saidmacrocyclic diterpene or its derivative or chemical analogue modulatesPKC activity, synthesis or enzyme turnover, said administration beingfor a time and under conditions sufficient to activate said virus.

The activated virus is then destroyed or removed by the host's ownimmune system and/or by anti-viral chemotherapy or by the effects of theagents themselves.

In a preferred embodiment, the method further comprises thesimultaneous, sequential or separate administration of an ancillaryagent which destroys or attenuates a replicating virus, in combinationwith the macrocyclic diterpene or chemical fraction. Thus, the inventioncontemplates the combined use of a macrocyclic diterpene or chemicalfraction which activates a latent virus to thereby cause the virus toundergo replication, and the use of an ancillary agent which eradicatesthe replicating virus.

Accordingly, in another aspect, the invention encompasses a method forthe treatment or prophylaxis of infection by a latent virus in asubject, said method comprising the simultaneous, sequential or separateadministration to said subject of a virus-activating effective amount ofa macrocyclic diterpene, or a chemical fraction comprising same from aplant of the family Euphorbiaceae or a derivative or chemical analogueof said macrocyclic diterpene having the structures as defined abovewherein said macrocyclic diterpene or its derivative or chemicalanalogue modulates PKC activity, synthesis or enzyme turnover, togetherwith a virus-destroying or -attenuating effective amount of an ancillaryagent which destroys or attenuates a replicating form of said virus,said administration being for a time and under conditions sufficient forsaid macrocyclic diterpene or chemical fraction to activate said virusto thereby cause the virus to undergo replication and for said ancillaryagent to destroy or attenuate said replicating virus.

In one embodiment, the latent virus is HIV. A range of HIV specificagents may be used for its destruction or attenuation, including, forexample, the agents described in Matsuhita et al., 2000. Preferred HIVspecific ancillary agents include, for example, nucleoside analoguessuch as combivir, epivir, hivid, retrovir, videx, zerat and zygen,non-nucleoside agents such as rescriptor, sustiva and viramune,adjunctive anti-retrovirals such as hydrea and droxa, and proteaseinhibitors such as agenerase, fortovase, crixivan, invirase, norvir andvirasept.

In another embodiment, the latent virus is EBV. Preferred EBV specificancillary agents are selected from ganciclovir (GVC) or3′-azido-3′deoxythymidine (AZT), as for example disclosed by Westphal etal., 2000.

In yet another embodiment, the latent virus is CMV. A preferred CMVspecific ancillary agent is cidofovir, as for example disclosed byPlatzbecker et al., 2001.

Particularly useful compounds include5,8,9,10,14-pentaacetoxy-3-benzoyloxy-15-hydroxypepluane (pepluane),derivatives of said pepluane, jatrophanes of Conformation II including2,3,5,7,15-pentaacetoxy-9-nicotinoyloxy-14-oxojatropha-6(17),11E-diene(jatrophane 1), derivatives of said jatrophane 1,2,5,7,8,9,14-hexaacetoxy-3-benzoyloxy-15-hydroxy-jatropha-6(17),11E-diene(atrophane 2), derivatives of said jatrophane 2,2,5,14-triacetoxy-3-benzoyloxy-8,15-dihydroxy-7-isobutyroyloxy-9-nicotinoyloxyjatropha-6(17),11E-diene(jatrophane 3), derivatives of said jatrophane 3,2,5,9,14-tetraacetoxy-3-benzoyloxy-8,15-dihydroxy-7-isobutyroyloxyjatropha-6(17),11E-diene)(jatrophane4), derivatives of said jatrophane 4,2,5,7,14-tetraacetoxy-3-benzoyloxy-8,15-dihydroxy-9-nicotinoyloxyjatropha-6(17),11E-diene(jatrophane 5), derivatives of said jatrophane 5,2,5,7,9,14-pentaacetoxy-3-benzoyloxy-8,15-dihydroxyjatropha-6(17),11E-diene(jatrophane 6), derivatives of said jatrophane 6, or pharmaceuticallyacceptable salts of these.

Even more particularly preferred compounds are angeloyl substitutedingenanes or derivatives thereof such as ingenol-3-angelate,20-deoxy-ingenol-3-angelate, 20-O-acetyl-ingenol-3-angelate, orderivatives of said angelates, or pharmaceutically acceptable salts ofthese.

Still a further aspect of the present invention contemplates a method ofassessing the suitability of a chemical agent from Euphorbiaceae for thepractice of the present invention. Numerical values are assigned tochemical agents including fractions comprising the chemical agents asset forth, for example, in Table A:

TABLE A Feature Value An ability to modulate PKC activity or effect +1An ability to induce bipolar dendritic activity +1 An ability todisplace phorbol dibutyrate from binding to PKC +1 An ability to inducerespiratory burst in leucocytes +1 An ability to stimulate phagocytosisin peripheral +1 blood mononuclear cells An ability to be derived from amember of the +1 Euphorbiaceae family Derived from E. peplus +3 Waterextractible from the sap of Euphorbia sp. +2 An ability to activatelatent virus +4 A lower tumor promotion activity than TPA/PMA +2

The value for each feature is referred to as the Index Value (I_(V)).

The sum of I_(V), i.e. ΣI_(V), provides a potency of agent (P_(A)) valueand this enables an analytical approach to screening and selectingcompounds from Euphorbiaceae useful in the practice of the presentinvention.

In one example, 20-acetyl-ingenol-3 angelate exhibits a P_(A)=ΣI_(V)=15.

Accordingly, another aspect of the present invention contemplates amethod for the treatment or prophylaxis of infection or colonization orpresence of a biological entity in a subject, said method comprisingadministration to said subject of a symptom-ameliorating effectiveamount of a macrocyclic diterpene obtainable from a Euphorbiaceae plantor its botanical or horticultural relative, said macrocyclic diterpenebeing selected from an ingenane, pepluane or jatrophane, or a derivativeor chemical analogue thereof, having the structure represented by anyone of the general formulae (I)-(V) as defined above and wherein saidchemical agent exhibits a potency of agent (P_(A)) of >10, wherein theP_(A)=ΣI_(V) where I_(V) is a numerical value associated with aparticular feature as listed below:

Feature Value An ability to modulate PKC activity or effect +1 Anability to induce bipolar dendritic activity +1 An ability to displacephorbol dibutyrate from binding to PKC +1 An ability to inducerespiratory burst in leucocytes +1 An ability to stimulate phagocytosisin peripheral +1 blood mononuclear cells An ability to be derived from amember of the +1 Euphorbiaceae family Derived from E. peplus +3 Waterextractible from the sap of Euphorbia sp. +2 Activity against latentvirus +4or pharmaceutically acceptable salts of these, said chemical agent beingadministered for a time and under conditions sufficient to ameliorate atleast one symptom caused by or associated with the biological entity.

Preferred compounds are selected from the list comprising:

-   5,8,9,10,14-pentaacetoxy-3-benzoyloxy-15-hydroxypepluane (pepluane);-   2,3,5,7,15-pentaacetoxy-9-nicotinoyloxy-14-oxojatropha-6(17),11E-diene    (jatrophane 1);-   2,5,7,8,9,14-hexaacetoxy-3-benzoyloxy-15-hydroxy-jatropha-6(17),11E-diene    (jatrophane 2);-   2,5,14-triacetoxy-3-benzoyloxy-8,15-dihydroxy-7-isobutyroyloxy-9-nicotinoyloxy    jatropha-6(17),11E-diene (jatrophane 3);-   2,5,9,14-tetraacetoxy-3-benzoyloxy-8,15-dihydroxy-7-isobutyroyloxy-jatropha-6(17),11E-diene    (jatrophane 4);-   2,5,7,14-tetraacetoxy-3-benzoyloxy-8,15-dihydroxy-9-nicotinoyloxy-jatropha-6(17),11E-diene    jatrophane 5);-   2,5,7,9,14-pentaacetoxy-3-benzoyloxy-8,15-dihydroxyjatropha-6(17),11    E-diene (jatrophane 6);-   20-O-acetyl-ingenol-3-angelate, derivatives of    20-O-acetyl-ingenol-3-angelate.-   20-hydroxy-ingenol-3-angelate, derivatives of    20-hydroxy-ingenol-3-angelate; and-   ingenol-3-angelate, derivatives of ingenol-3-angelate.

Preferably, the biological entity is a microorganism, virus, yeast,fungus, insect, arachnid or Plasmodium sp.

Reference herein to a subject includes a human, primate, livestockanimal (e.g. sheep, cow, horse, pig, goat, donkey), laboratory testanimal (e.g. mouse, rat, guinea pig, hamster), companion animal (e.g.dog, cat) or avian species such as poultry birds (e.g. chicken, ducks,turkeys, geese) or game birds (e.g. arid ducks, pheasants).

The preferred subject is a human or primate or laboratory test animal.

The most preferred subject is a human.

The ability to assign numerical values to certain characteristicsenables data processing means to assess the likely usefulness of aparticular compound or group of compounds forming a chemical agent.

The assessment of the suitability of a compound or group of compoundsfor the practice of the present invention is suitably facilitated withthe assistance of a computer programmed with software, which inter aliaadds index values (I_(V)) for at least two features associated with thecompound(s) to provide a potency value (P_(A)) corresponding to theeffectiveness of the compound(s) for treating or preventing infection orcolonization or presence of a biological entity in a subject. Thecompound features can be selected from:

-   (a) the ability to modulate PKC activity or effect;-   (b) the ability to induce bipolar dendritic activity;-   (c) the ability to be derived from a member of the Euphorbiaceae    family;-   (d) the ability to be derived from E. peplus;-   (e) the ability to be water extractable from the sap of a Euphorbia    species; or-   (f) the ability to activate latent virus;-   (g) less tumor promoting capacity than TPA or PMA.

Accordingly, in accordance with the present invention, index values forsuch features are stored in a machine-readable storage medium, which iscapable of processing the data to provide a potency value for a compoundor group of compounds of interest.

Thus, in another aspect, the invention contemplates a computer programproduct for assessing the likely usefulness of a candidate compound orgroup of compounds for treating or preventing infection or colonizationor presence of a biological entity in a subject, said productcomprising:

-   (1) code that receives as input index values for at least two    features associated with said compound(s), wherein said features are    selected from:    -   (a) the ability to modulate PKC activity or effect;    -   (b) the ability to induce bipolar dendritic activity;    -   (c) the ability to be derived from a member of the Euphorbiaceae        family;    -   (d) the ability to be derived from E. peplus;    -   (e) the ability to be water extractable from the sap of a        Euphorbia species; or    -   (f) the ability to activate latent virus;    -   (g) less tumor promoting capacity than TPA or PMA;-   (4) code that adds said index values to provide a sum corresponding    to a potency value for said compound(s); and-   (5) a computer readable medium that stores the codes.

In a preferred embodiment, the computer program product comprises codethat assigns an index value for each feature of a compound or group ofcompounds. In an especially preferred embodiment, index values areassigned as set forth in Table A above.

In a related aspect, the invention extends to a computer for assessingthe likely usefulness of a candidate compound or group of compounds fortreating or preventing infection or colonization or presence of abiological entity in a subject, wherein said computer comprises:

-   (1) a machine-readable data storage medium comprising a data storage    material encoded with machine-readable data, wherein said    machine-readable data comprise index values for at least two    features associated with said compound(s), wherein said features are    selected from:    -   (a) the ability to modulate PKC activity or effect;    -   (b) the ability to induce bipolar dendritic activity;    -   (c) the ability to be derived from a member of the Euphorbiaceae        family;    -   (d) the ability to be derived from E. peplus;    -   (e) the ability to be water extractable from the sap of a        Euphorbia species; or    -   (f) the ability to activate latent virus;    -   (g) less tumor promoting capacity than TPA or PMA;-   (2) a working memory for storing instructions for processing said    machine-readable data;-   (3) a central-processing unit coupled to said working memory and to    said machine-readable data storage medium, for processing said    machine readable data to provide a sum of said index values    corresponding to a potency value for said compound(s); and-   (4) an output hardware coupled to said central processing unit, for    receiving said potency value.

A version of these embodiments is presented in FIG. 8, which shows asystem 10 including a computer 11 comprising a central processing unit(“CPU”) 20, a working memory 22 which may be, e.g. RAM (random-accessmemory) or “core” memory, mass storage memory 24 (such as one or moredisk drives or CD-ROM drives), one or more cathode-ray tube (“CRT”)display terminals 26, one or more keyboards 28, one or more input lines30, and one or more output lines 40, all of which are interconnected bya conventional bidirectional system bus 50.

Input hardware 36, coupled to computer 11 by input lines 30, may beimplemented in a variety of ways. For example, machine-readable data ofthis invention may be inputted via the use of a modem or modems 32connected by a telephone line or dedicated data line 34. Alternativelyor additionally, the input hardware 36 may comprise CD. Alternatively,ROM drives or disk drives 24 in conjunction with display terminal 26,keyboard 28 may also be used as an input device.

Output hardware 46, coupled to computer 11 by output lines 40, maysimilarly be implemented by conventional devices. By way of example,output hardware 46 may include CRT display terminal 26 for displaying asynthetic polynucleotide sequence or a synthetic polypeptide sequence asdescribed herein. Output hardware might also include a printer 42, sothat hard copy output may be produced, or a disk drive 24, to storesystem output for later use.

In operation, CPU 20 coordinates the use of the various input and outputdevices 36,46 coordinates data accesses from mass storage 24 andaccesses to and from working memory 22, and determines the sequence ofdata processing steps. A number of programs may be used to process themachine readable data of this invention. Exemplary programs may use forexample the following steps:

-   (1) inputting input index values for at least two features    associated with said compound(s), wherein said features are selected    from:    -   (a) the ability to modulate PKC activity or effect;    -   (b) the ability to induce bipolar dendritic activity;    -   (c) the ability to be derived from a member of the Euphorbiaceae        family;    -   (d) the ability to be derived from E. peplus;    -   (e) the ability to be water extractable from the sap of a        Euphorbia species; or    -   (f) the ability to activate latent virus;    -   (g) less tumor promoting capacity than TPA or PMA;-   (2) adding the index values for said features to provide a potency    value for said compound(s); and (3) outputting said potency value.

FIG. 9 shows a cross section of a magnetic data storage medium 100 whichcan be encoded with machine readable data, or set of instructions, fordesigning a synthetic molecule of the invention, which can be carriedout by a system such as system 10 of FIG. 10. Medium 100 can be aconventional floppy diskette or hard disk, having a suitable substrate101, which may be conventional, and a suitable coating 102, which may beconventional, on one or both sides, containing magnetic domains (notvisible) whose polarity or orientation can be altered magnetically.Medium 100 may also have an opening (not shown) for receiving thespindle of a disk drive or other data storage device 24. The magneticdomains of coating 102 of medium 100 are polarized or oriented so as toencode in manner which may be conventional, machine readable data suchas that described herein, for execution by a system such as system 10 ofFIG. 8.

FIG. 10 shows a cross section of an optically readable data storagemedium 110 which also can be encoded with such a machine-readable data,or set of instructions, for designing a synthetic molecule of theinvention, which can be carried out by a system such as system 10 ofFIG. 8. Medium 110 can be a conventional compact disk read only memory(CD-ROM) or a rewritable medium such as a magneto-optical disk, which isoptically readable and magneto-optically writable. Medium 100 preferablyhas a suitable substrate 111, which may be conventional, and a suitablecoating 112, which may be conventional, usually of one side of substrate111.

In the case of CD-ROM, as is well known, coating 112 is reflective andis impressed with a plurality of pits 113 to encode the machine-readabledata. The arrangement of pits is read by reflecting laser light off thesurface of coating 112. A protective coating 114, which preferably issubstantially transparent, is provided on top of coating 112.

In the case of a magneto-optical disk, as is well known, coating 112 hasno pits 113, but has a plurality of magnetic domains whose polarity ororientation can be changed magnetically when heated above a certaintemperature, as by a laser (not shown). The orientation of the domainscan be read by measuring the polarisation of laser light reflected fromcoating 112. The arrangement of the domains encodes the data asdescribed above.

The present invention further extends to pharmaceutical compositionsuseful in treating a pathogenic infection. In this regard, the chemicalagents of the invention can be used as actives for the treatment orprophylaxis of a condition associated with the presence of a biologicalentity or part thereof or toxin or venom therefrom or a genetic eventcaused thereby in a subject. The chemical agents can be administered toa patient either by themselves, or in pharmaceutical compositions wherethey are mixed with a suitable pharmaceutically acceptable carrier.

Accordingly, the invention also provides a composition for treatmentand/or prophylaxis of a condition associated with the presence of abiological entity or part thereof or toxin or venom therefrom or agenetic event caused thereby in a subject, comprising one or morechemical agents of the invention, together with a pharmaceuticallyacceptable carrier and/or diluent.

Depending on the specific conditions being treated, chemical agents maybe formulated and administered systemically or locally. Techniques forformulation and administration may be found in “Remington'sPharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latestedition. Suitable routes may, for example, include oral, rectal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections. For injection,the chemical agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks' solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art. Intra-muscular and subcutaneous injection isappropriate, for example, for administration of immunomodulatorycompositions and vaccines.

The chemical agents can be formulated readily using pharmaceuticallyacceptable carriers well known in the art into dosages suitable for oraladministration. Such carriers enable the compounds of the invention tobe formulated in dosage forms such as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient to be treated. These carriers may be selected from sugars,starches, cellulose and its derivatives, malt, gelatine, talc, calciumsulphate, vegetable oils, synthetic oils, polyols, alginic acid,phosphate buffered solutions, emulsifiers, isotonic saline, andpyrogen-free water.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve their intended purpose. The dose of agentadministered to a patient should be sufficient to effect a beneficialresponse in the patient over time such as a reduction in the symptomsassociated with the presence of a biological entity or part thereof ortoxin or venom therefrom or a genetic event caused thereby in a subject.The quantity of the agent(s) to be administered may depend on thesubject to be treated inclusive of the age, sex, weight and generalhealth condition thereof. In this regard, precise amounts of theagent(s) for administration will depend on the judgement of thepractitioner. In determining the effective amount of the chemical agentto be administered in the treatment or prophylaxis of a conditionassociated with the biological entity, the physician may evaluate tissueor fluid levels of the biological entity, and progression of thedisorder. In any event, those of skill in the art may readily determinesuitable dosages of the chemical agents of the invention.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as., for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinyl-pyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Such compositions may beprepared by any of the methods of pharmacy but all methods include thestep of bringing into association one or more chemical agents asdescribed above with the carrier which constitutes one or more necessaryingredients. In general, the pharmaceutical compositions of the presentinvention may be manufactured in a manner that is itself known, e.g. bymeans of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilisingprocesses.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added.

Dosage forms of the chemical agents of the invention may also includeinjecting or implanting controlled releasing devices designedspecifically for this purpose or other forms of implants modified to actadditionally in this fashion. Controlled release of an agent of theinvention may be effected by coating the same, for example, withhydrophobic polymers including acrylic resins, waxes, higher aliphaticalcohols, polylactic and polyglycolic acids and certain cellulosederivatives such as hydroxypropylmethyl cellulose. In addition,controlled release may be effected by using other polymer matrices,liposomes and/or microspheres.

Chemical agents of the invention may be provided as salts withpharmaceutically compatible counterions. Pharmaceutically compatiblesalts may be formed with many acids, including but not limited tohydrochloric, sulphuric, acetic, lactic, tartaric, malic, succinic, etc.Salts tend to be more soluble in aqueous or other protonic solvents thatare the corresponding free base forms.

For any chemical agent used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating concentration range that includes the IC50 asdetermined in cell culture (e.g. the concentration of a test agent,which achieves a half-maximal inhibition of infection or colonization orpresence of a biological entity). Such information can be used to moreaccurately determine useful doses in humans.

Toxicity and therapeutic efficacy of such chemical agents can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g. for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Compounds that exhibit large therapeutic indices arepreferred. The data obtained from these cell culture assays and animalstudies can be used in formulating a range of dosages for use in humans.The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition (see for exampleFingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.1 p 1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active agent which are sufficient to maintainsymptom-ameliorating effects. Usual patient dosages for systemicadministration range from 1-2000 mg/day, commonly from 1-250 mg/day, andtypically from 10-150 mg/day. Stated in terms of patient body weight,usual dosages range from 0.02-25 mg/kg/day, commonly from 0.02-3mg/kg/day, typically from 0.2-1.5 mg/kg/day. Stated in terms of patientbody surface areas, usual dosages range from 0.5-1200 mg/m²/day,commonly from 0.5-150 mg/m²/day, typically from 5-100 mg/m²/day.

Alternately, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto a tissue, often in a depot or sustained release formulation.Furthermore, one may administer the drug in a targeted drug deliverysystem, for example, in a liposome coated with tissue-specific antibody.The liposomes will be targeted to and taken up selectively by thetissue. In cases of local administration or selective uptake, theeffective local concentration of the agent may not be related to plasmaconcentration.

The chemical agents of the invention can also be delivered topically.For topical administration, a composition containing between 0.001-5% ormore chemical agent is generally suitable. Regions for topicaladministration include the skin surface and also mucous membrane tissuesof the vagina, rectum, nose, mouth, and throat. Compositions for topicaladministration via the skin and mucous membranes should not give rise tosigns of irritation, such as swelling or redness.

The topical composition may include a pharmaceutically acceptablecarrier adapted for topical administration. Thus, the composition maytake the form of a suspension, solution, ointment, lotion, sexuallubricant, cream, foam, aerosol, spray, suppository, implant, inhalant,tablet, capsule, dry powder, syrup, balm or lozenge, for example.Methods for preparing such compositions are well known in thepharmaceutical industry.

In one embodiment, the topical composition is administered topically toa subject, e.g. by the direct laying on or spreading of the compositionon the epidermal or epithelial tissue of the subject, or transdermallyvia a “patch”. Such compositions include, for example, lotions, creams,solutions, gels and solids. Suitable carriers for topical administrationpreferably remain in place on the skin as a continuous film, and resistbeing removed by perspiration or immersion in water. Generally, thecarrier is organic in nature and capable of having dispersed ordissolved therein a chemical agent of the invention. The carrier mayinclude pharmaceutically-acceptable emolients, emulsifiers, thickeningagents, solvents and the like.

The invention also features a process for separating macrocyclicditerpenes from a biomass containing same, said process comprisingcontacting the biomass with an aqueous solvent for a time and underconditions sufficient to extract the macrocyclic diterpenes into saidsolvent.

The aqueous solvent is preferably water.

Suitably, the biomass is derived from a plant, which is preferably amember of the Euphorbiaceae family of plants or botanical orhorticultural relatives of such plants. Matter from the plant (e.g.foliage, stems, roots, seeds, bark, etc.) is preferably cut, maceratedor mulched to increase the surface area of the plant matter for aqueousextraction of the macrocyclic diterpenes.

The process preferably further comprises adsorbing the macrocyclicditerpenes to a non-ionic adsorbent, which is suitably a non-ionicporous synthetic adsorbent. Among the non-ionic porous syntheticadsorbents that can be used for the purposes of the present inventioninclude, but are not restricted to, aromatic copolymers mainly composedof styrene and divinylbenzene, and methacrylic copolymers mainlycomposed of monomethacrylate and dimethacrylate. Such non-ionic poroussynthetic adsorbents which comprise, as the basic structure, aromaticcopolymers mainly composed of styrene and divinylbenzene include, forexample, Diaion HP10, HP20, HP21, HP30, HP40, HP50, SP850, and SP205(trade names: Mitsubishi Chemical Corp.), and Amberlite XAD-2, XAD4,(trade names: Rohm and Haas Co.). Examples of non-ionic porous syntheticadsorbent which comprise, as the basic structure, methacrylic copolymermainly composed of monomethacrylate and dimethacrylate are Diaion HP2MG,Amberlite XAD-7, XAD-8 and XAD-16 and others.

Preferably, the process further comprises eluting macrocyclic diterpenesfrom the non-ionic adsorbent with water and water-soluble organicsolvent(s).

The treatment may be conducted by a batch method using water andwater-soluble organic solvent(s) which dissolve macrocyclic diterpenes,or may also be conducted continuously or in batch using a columnchromatography method.

Examples of a water-soluble organic solvent which may be used in thepresent invention are alcohols such as methanol, ethanol, n-propylalcohol, isopropyl alcohol, and tert-butanol, ethers such as dioxane andtetrahydrofuran, ketones such as acetone, amides such asdimethylformamide, sulfur-containing compounds such asdimethylsulfoxide. Two or more of such organic solvents may be mixed foruse. In addition, solvents less soluble in water, for example, alcoholssuch as n-butanol, esters such as methyl formate and methyl acetate, andketones such as methyl ethyl ketone may also be used to the extent thatit does not separate during development. Particularly preferredwater-soluble organic solvents are alcohols, in particular, methanol,ethanol, propyl alcohol, and the like. Furthermore, different kinds ofsolvent may also be used sequentially for development.

Macrocyclic diterpenes can be further purified using media andtechniques which separate compounds on the basis of molecular sizeand/or polarity. In a preferred embodiment of this type, the macrocyclicditerpenes are separated using Sephadex HL-20 resin and preferably usingwater and water-soluble organic solvent(s) for development.

The present invention is further described by the following non-limitingExamples.

EXAMPLE 1 PKC Activation: Kinase Activity of PKC as Measured by EnzymeAssay

Preparation of Chemical fractions From E. peplus

Sap from E. peplus plants was collected, stored at −20° C., thawed andstored at 4° C. for 1 week prior to use. The H fraction was preparedfrom frozen sap by thin layer chromatography (TLC) as described inInternational Patent Application No. PCT/AU98/00656 and was stored asdried silica-associated material at 4° C. This material was enriched injatrophanes and pepluanes. One to two months prior to use, the materialwas dissolved in ethylene glycol dimethyl ether (DME) and stored at 4°C. The concentrations were determined from the dry weight of thematerial. For PKC assays, crude sap (PEP001) and the PEP004 fraction wasether extracted twice to produce an ether-soluble fraction enriched inditerpenes, namely, ingenanes, jatrophanes and pepluanes. The remainingwater soluble fraction was also used. An ingenane fraction was preparedfrom the ether-soluble extract by TLC as described in InternationalPatent Application No. PCT/AU98/00656, which corresponds to U.S. Pat.No. 6,432,452, the contents of which are incorporated by reference.

PKC Assay

The conventional and novel protein kinase C (PKC) isoforms, in theirunstimulated state, are inactive as kinases. The C1 domain of these PKCscontains an autoinhibitory, pseudosubstrate site that binds to thesubstrate site (C4 domain) and inactivates the kinase functionality ofthe protein. Activation of PKC results from binding of diacylglycerol(DAG) to the C1 domain, which, via multiple phosphorylation events andconformational changes to the protein, ultimately leads to the releaseof PKC autoinhibition. TPA and other related compounds have been shownto bind to the C1 domain of various PKC isoforms and presumably bysimilar means as DAG, lead to their activation.

The kinase activity of rat brain PKC (Promega) was determined using thePeptag™ Non-Radioactive Protein Kinase Kit (Promega). Using agarose gelelectrophoresis the technique visualises the opposing electrostaticcharge of a fluorescently labeled peptide (PLSRTLSVAAK) compared to thephosphorylated version of the same peptide.

The results of an assay of PKC with the fluorescent substrate (PepTag)are shown in FIG. 1. The reaction mixture was separated by gelelectrophoresis, showing migration of the unreacted substrate (a) to theanode (top), and the product (b), which is more negatively chargedbecause of phosphorylation by PKC, moving towards the cathode (bottom).The positive control activator (phosphatidyl serine) supplied by themanufacturer (lane 2) showed strong activation compared with PKC andsubstrate alone (lane 1). Various dilutions of TPA also showedactivation of PKC (lanes 3, 4 and 5).

An ether extract of E. peplus sap, reconstituted in dimethoxyethane(DME) and incubated with PKC at a final dilution of 1 in 5 relative tothe sap, gave a significant level of action (lane 7), as did the crudesap itself (lane 9). In the latter case, however, both the substrate andproduct (band c, lane 9) were found further towards the cathode. Thisresult was interpreted as being due to a carboxypeptidase activity inthe crude sap, cleaving the C-terminal, positively-charged lysine fromthe substrate peptide. This was confirmed by the finding that theaqueous layer from ether extraction had minimal PKC-activating ability,but altered migration of the substrate in the same way as the crude sap(lane 8). DME itself had no activity (lane 10).

FIG. 2 shows the results of testing fractionated materialssimultaneously with negative (lane 1) and positive controls (lane 2).Fraction H (mixture of jatrophanes and pepluanes) showed a low activity(lane 3), seen as a halo of product (arrow) moving away from theunreacted substrate. A similar result was found for the ingenanefraction (lane 4).

All of the E. peplus fractions are tested for activation of all theavailable protein kinase enzymes using the peptide-based fluorescent tagtest described above. The isoenzymes available for this experiment(Panevera) were α, β1, β11, γ, δ, ε, η and ζ.

Essentially, the kinase activity of the PKC sample was assessed beforestimulation (Negative Control) and after stimulation with PEP001,phosphatidyl serine (an acid-lipid, known to activate PKC, provided byPromega; Positive Control) and TPA (20 μg/mL). The results presented inFIG. 3 indicate that PEP001, at dilutions of 1:125 and 1:500, activatesPKC to a similar level as phosphatidyl serine (200 μg/mL) and to agreater level than TPA (20 μg/mL). From this experiment, it is clearthat the PEP001 activates PKC.

EXAMPLE 2 PKC Activation: Translocation of PKC

Activation of PKC can also be demonstrated by a simple fluorescencemicroscopy-based assay. Upon activation, PKC is known to translocatefrom the cytoplasm to the plasma membrane of the cell. By fusing PKCenzymes to the green fluorescent protein (GFP) or enhanced GFP (EGFP),activation of the PKC can be detected by the movement of diffusecytoplasmic GFP to a ring of fluorescence associated with the plasmamembrane. Using this assay, crude E. peplus extract has been shown toactivate PKCβ and PKCγ.

MM96L cells were first transfected using a commercially-available kit(Qiagen Effectine Transfection Kit) with a PKC-GFP expression vector(Clontech; http://www.clontech.com/gfp/) and allowed to produce thePKC-GFP protein for 24 hr. The cells were then treated with crude E.peplus extract and TPA and observed under a fluorescent microscope (488nm excitation). Two controls were used—no DNA, which allows for theidentification of non-transfected cells, and no drugs, which allows forthe calculation of transfection efficiency and the identification oftransfected cells without PKC activation. pPKCβ-EGFP and pPKCγ-EGFP weretested, and crude E. peplus extract was shown to induce movement of thefluorescence from the cytosol to the plasma membrane, indicating thatcrude E. peplus extract activated these PKC enzymes. The results areillustrated in FIGS. 4A and 4B, which respectively show expression ofPKCβ in the absence of any drug and after exposure to crude E. peplusextract for 2 hr.

In another experiment, translocation of individual PKC isoforms wasobserved using fluorescence microscopy and used as an indication ofactivation by PEP003 and PEP005.

Five EGFP-PKC isoforms (Clontech) were available for this experiment,enabling the screening of the three predominant PKC families (i.e.classical, novel and a typical PKCs). The members of the various PKCfamilies are α, β, and γ (classical), θ (novel) and ζ (atypical).

HeLa cells were plated out in a 24-well plate containing coverslips andtransfected with PKC isoforms fused to EGFP, using a commerciallyavailable effectine-transfection kit (QIAGEN, Pty. Ltd.). Cells wereexposed to the transfection reagents for 16-24 hr. Subsequently,transfected cells were treated for one hour with TPA (100 ng/mL),bryostatin-1 (5 pg/mL), PEP003 (2.25 μg/mL; 5 μM) or PEP005 (670 μg/mL)1.5 μM). Following treatment, cells were fixed on coverslips and mountedon glass slides. The slides were subsequently examined visually byfluorescence microscopy, photographed, and over 150 cells werecounted/treatment/PKC isoform. Counted cells were classified accordingto the localisation of the PKC-EGFP fluorescence as either cytoplasmicor plasma membrane using ImagePro™ 4.1 (FIG. 5). Several cells alsoshowed localisation to the Golgi, or similarly located cellularstructure (FIG. 5). The number of these cells was also counted. Resultsare presented as an average and standard deviation of percentages ofcells (Table 1).

The results presented in FIG. 6 show that PKC α, β and γ aretranslocated from the cytoplasm to the plasma membrane in response totreatment with PEP003, PEP005 and TPA but not with bryostatin-1. Asexpected, the diacylglycerol-independent PKCζ is not translocated inresponse to any treatment. PKCθ is translocated in response to PEP003,TPA and bryostatin-1, however, PEP005 does not induce any change in theisoenzymes localization. The results also show that treatment of PKCαand γ transfected cells with TPA, PEP003 and PEP005 leads to an increasein the number of cells displaying Golgi-like fluorescence. PKCβtransfected HeLa cells treated with TPA also show an increase inGolgi-like fluorescence. In contrast, treatment with PEP005 andbryostatin-1 decreases the number of cells with PKCβ concentrated in theGolgi. The number of PKCθ transfected HeLa cells with Golgi-likelocalization is increased in response to all treatments.

The above results indicate that PEP003 and PEP005 induce translocationof the classical and novel PKC isoforms tested, suggesting that thesecompounds activate members of the classical and novel PKC families. TPA,Bryostatin-1, PEP003 and PEP005 fail to induce translocation of PKCζ,suggesting that PEP003 and PEP005 do not activate members of the atypical PKC family. Furthermore, TPA, Bryostatin-1, PEP003 and PEP005display differences in their ability to induce translocation of thespecific PKC isoforms to the plasma membrane and/or Golgi. Thesedifferences may play a role in determining the different biologicalactions of these compounds.

EXAMPLE 3 Binding of Compounds to PKC

A competition assay was performed to determine whether the diterpeneesters of the instant invention bind to the phorbol ester binding siteof PKC. This competition assay showed that 23 μg/mL PEP003displaced >90% [3H]-phorbol dibutyrate from binding to rat brainhomogenate, used as a source of PKC (Gonzalez et al., 1999). Thisbinding was not blocked by co-incubation with bisindolylmaleimide. Theseresults show that PEP003 binds to the phorbol ester binding site of PKC,and bisindolylmaleimide does not.

EXAMPLE 4 Activation of Latent HIV Infection

The use of highly active anti-retroviral therapy such as combinations ofreverse transcriptase inhibitors and protease inhibitors (HAART) hassignificantly prolonged the life of individuals infected with HIV.However, the regimen is very burdensome, requiring strict adherence toprevent a recurrence of viraemia. Long-lived cells capable of activelytranscribing virus, such as CD4⁺ cells, act as a major latent reservoirand enable the virus to avoid anti-retroviral chemotherapy or immunesystem surveillance. There is, therefore, an urgent need to find anagent which activates latent virus from the infected cells. Activatedvirus could then be killed by aggressive anti-retroviral chemotherapyand it has been hypothesized that immune system surveillance could alsobe improved under these conditions. Such an agent could have utility inother disease states in which virus is sequestered in infected cells,e.g. herpes infections. Anti-cancer agents have been widely investigatedas potential anti-HIV agents. Several PKC activators have been shown toactivate latent retroviruses. For example, PMA has been shown toactivate latent HIV in monocytes (Tobiume et al., 1998). However, PMA isa known tumor promoter.

A latently HIV-1 infected cell line (U1), derived from the promonocyticcell line U937 after infection with HIV-1 LAI strain, was used in theseexperiments. In the absence of activation, no or little virus (measuredas p24 production) is produced by the U1 cell line. Phorbol esters areknown to activate virus production from these cells (Tobiume et al.,1998) and so TPA/PMA was used as a positive control in theseexperiments.

U1 cells were cultured in RPM1-1640 medium supplemented with 10% fetalbovine serum, 10⁵ cells/mL were cultured for 20 hr in the presence andabsence of various concentrations of either the phorbol ester TPA orcrude E. peplus sap (PEP001) or PEP004 (H1) derived therefrom.Supernatants were collected and viral replication monitored bydetermination of the amounts of HIV p24 gag protein in the culturesupernatants by ELISA, using a NEN Life Science HIV-1 p24 ELISA kit. p24values were calculated from OD values using a standard curve.

TPA, the crude sap (PEP001) from E. peplus and the PEP004 fraction allactivated HIV from U1 cells, as illustrated in FIG. 7. The crude sap(PEP001) was 50 times less active than TPA. The PEP004 fraction was 1000times less active than TPA.

EXAMPLE 5 Lytic HIV Activity Inhibited by PEP003 and PEP004

The human immunodeficiency virus (HIV), a retrovirus, is the cause ofthe fourth greatest killing disease in the world, infecting more than 36million people. A number of anti-retroviral compounds have been approvedfor clinical use, but many HIV strains have developed resistance tothese drugs. There is clear and immediate need for new anti-retroviralcompounds.

Experiments were conducted to assess the effect of the compounds of theinstant invention on HIV-1 replication in acutely infected T cells.Peripheral blood mononuclear cells (PBMC) were obtained from non-HIV-1,non-HIV-2, non-Hepatitis B/C infected donors, stimulated withphytohemagglutinin-M and grown in culture media supplemented with 10U/mL interleukin-2. The activated PBMC were infected with 10 g (LowTitre) and 100 ng (High Titre) of CA-p24 equivalents of the HIV-strainpNL4-3. Cells were infected for two hr after which, the virus wasremoved and the cells were washed with culture media. Equivalent numbersof cells were seeded into 24 well plates and compounds were added to thecultured cells that included: TPA at 8 nM and 80 nM, Ingenol at 280 nM,PEP003 at 500 nM, 50 nM and 5 nM, or PEP004 at dilutions of 1×10⁴ and1×10⁵ from the stock (final concentrations). In addition, uninfectedactivated PBMC were grown in the presence of TPA (80 nM), Ingenol (280nM), PEP003 (500 nM) and PEP004 (1×10⁴ dilution). Other cultures wereneither infected nor treated with any compound, or infected but nottreated with any compound. Supernatant was removed from each culture atday 0, 3, 7, and 10. The amount of HIV-1 CA-p24 was determined using acommercially available ELISA assay. Three independent experiments wereperformed.

The data presented in FIGS. 8A-8D show that PEP003 reduced virusreplication kinetics in a dose-dependent manner. PEP003 atconcentrations of 500 nM, 50 nM and 5 nM reduced the replication rate byapproximately 99.9%, 95% and 47%, respectively, relative to theuntreated, infected cells. PEP004 at dilutions of 1×10 ⁴ and 1×10⁵reduced the replication rate by approximately 66% and 15%, respectively.Viral load seemed to alter these results slightly, as higher initialinoculums of virus reduced the total inhibition of PEP003 at 500 nM or50 nM to approximately 97% (t-test; p<0.001) or 88% (t-test; p<0.074),respectively. The control compounds Ingenol (2.8 μM) and TPA (80 nM or 8nM) reduced HIV-1 replication rates by approximately 35%, 98% and 38%,respectively.

EXAMPLE 6 Enhancement of the Cytomegalovirus Promoter Activity as aMethod for Improving Gene Therapy

Viruses and viral promoters especially adenovirus and CMV are used todeliver gene therapy in a range of human disease conditions. Geneexpression and, hence, therapeutic effect will be enhanced if thepromoters driving their transcription can be activated further by anagent.

Human melanoma cells were infected with ten-fold dilutions of adenovirus5 in culture, treated with dilutions of PEP005, PEP006, PEP008 andPEP010 and adenovirus replication determined 2 days later byimmunhistochemical detection of virus-replicating cells. Virusreplication (enumerated as the number of stained cells followingsuccessive incubations with adenovirus antibody, peroxidase-conjugatedprotein A and peroxidase substrate) was increased by 344% with 67 ng/mLPEP005, 256% with 295 ng/mL PEP006, 248% with 226 ng/mL PEP008 and 147%with 67.5 ng/mL PEP010.

The CMV promoter is commonly used to activate the transcription of genesin constructs transfected into a variety of cells, due to its strongtranscriptional activity in a variety of human cell types. The abilityof TPA to increase this activity has been demonstrated in cellsundergoing non-productive infection with an adenovirus construct(Christenson et al., 1999), thus raising the possibility of increasingthe production of a therapeutic protein encoded by a similar construct.

Human melanoma cells (MM96L; 50,000 per microtiter well) were treatedwith TPA or dilutions of crude E. peplus sap, infected with a 1/20dilution of a pool of adenovirus-5 expressing β-galactosidase driven bythe CMV promoter. After incubation for 20 hr, the wells were washed with3×with PBS, 50 μL of chlorophenol red galactoside (GPRG) substratesolution added and the absorbance at 540 nm read after 90 min. Theinventors found TPA (100 ng/mL) and crude E. peplus sap (diluted 1 in10,000) both induced the CMV promoter activity by >3-fold.

EXAMPLE 7 Activation of Innate Immune Responses: Induction of NeutrophilInvasion in Skin

Neutrophils represent about 70% of peripheral white blood cells inhumans and play a pivotal role in inflammation and the innate defenseagainst disease (Mollinedo, 1999). Upon activation, neutrophils releasesuperoxide radicals and granules containing a variety of enzymes andother compounds. These secretions are able to destroy invadingpathogens, but also result in inflammation and associated tissue damage.

The inventors found that E. peplus sap causes accumulation ofneutrophils at the site of application, showing that E. peplus sap iscapable of recruiting neutrophils. A mixture of active diterpenesobtained as an ether extract from E. peplus sap was applied (2 μL of 100mg/mL in ethanol) on the skin of a nu/nu mouse. After 24 hr, the animalwas sacrificed and the skin fixed in 10% formalin for sectioning andhematoxylin/eosin staining. As shown in FIGS. 9A and 9B, control skinshowed normal skin structure with few infiltrating monocytes. Thetreated skin showed large numbers of infiltrating neutrophils,characterized by their polymorphic nuclei. There was no evidence ofgross damage to the skin.

EXAMPLE 8 Neutrophil Infiltration Activity

Basal cell carcinoma (BCC) is the most common cancer in the Caucasianpopulation, with the highest annual incidence globally having beenrecorded in Australia (Miller et al., 1994, Marks et al., 1993). Newdevelopments have begun looking at treating non-melanoma skin cancer(NMSC) using topical therapies. The essence of this therapy may relyupon the induction of an inflammatory response with infiltration ofleucocytes, in particular neutrophils.

To assess whether the compounds of the invention induce neutrophilinfiltration, an experiment was designed on C57BL/6J mice. Twenty-fourmice were divided into six groups of four mice per group. In three ofthese groups the mice had a B16 melanoma injected s.c. (2 sites permouse, 5×10⁵ cells/site), that was left to grow for 8 days toapproximate tumor sizes of 5-8 mm in diameter. A single application ofone of all three compounds was then applied to the tumor or to normalskin. Each compound was applied on two groups of mice, one with tumorand 1 without tumor. The three compounds were PEP010 (2 μL; 150 mM) in10 μL of isopropanol gel (isopropyl alcohol 25% (w/w), propyl alcohol25% (w/w)) (vehicle), PEP009 (2 μL of stock) in 10 μL of vehicle orvehicle alone as a control. One mouse from each group was thensacrificed at either 4 hr, 24 hr, 48 hr or 144 hr post singleapplication of compound and then tissue excised and sections preparedfor histology.

The results at 4 hr show only minimal response with 1+ patchyneutrophils for both PEP010 on B16 tumor and PEP009 on normal skin and2+ neutrophils present for PEP009 on B16 tumor (Table 2). At 24 hr,there are no neutrophils present in the control groups with vehiclealone but a 4+ neutrophil infiltration with PEP010 and PEP009application, on both tumor and normal skin (FIGS. 10A and 10B). Inaddition, 60-85% of the superficial tumor cells were apoptotic ornecrotic in the B 16 groups. At 48 hr, there was a similar pattern witha 4+ neutrophil presence with PEP010 and PEP009 application while thecontrol groups showed an absence of neutrophils (FIGS. 10A and 10B).Along with the tumor cell necrosis and apoptosis, there is also evidenceof some neutrophil breakdown at the 48 hour interval. The 144 hour groupshowed a lack of neutrophils in the control group and a presence of 2-4+neutrophils, which were mostly now degenerate in the PEP010 and PEP009groups. There was extensive necrosis of tumor and skin, and clear signsof granulation tissue and early repair.

This study shows that the PEP010 and PEP009 induce a marked inflammatoryinfiltrate of neutrophils as compared to vehicle alone and this influxof polymorphonuclear cells may be significant in altering the growth ofcertain skin cancers.

EXAMPLE 9 Activation of Innate Immune Responses: Induction of aRespiratory Burst in Peripheral Blood Mononuclear Cells

Monocytes/macrophages are blood-borne and tissue cells which are usuallyactivated by T lymphocytes and antibodies. Upon activation, they areable to phagocytose pathogens, release superoxide radicals and are animportant source of cytokines. Crude E. peplus extract was shown to beable to induce the release of superoxide radicals by use of afluorescence-activated cell sorting (FACS)-based method, in whichsuperoxide radicals are detected by the dye dihydroethidium. Inaddition, phagocytic activity was activated by E. peplus, as shown byincreased uptake of nitroblue tetrazolium and adherence to plastic wasincreased by E. peplus; this is believed to indicate activation anddifferentiation of macrophages.

Human peripheral blood mononuclear cells (PBMC) prepared by standardFicoll separation comprise approximately 5% monocytes. PBMC wereincubated with dihydroethidium, a reduced form of the dye which becomesfluorescent when oxidized by a respiratory burst, then treated in 10%FCS-RPMI 1640 at 37° C. for 15 min with crude E. peplus extract diluted1/1000 or 100 ng/mL TPA and analyzed by flow cytometry usingconventional methods (Handbook of Flow Cytometry Methods, p. 151). Themean channel numbers for fluorescence were 618 (controls) and 818 (E.peplus extract diluted 1/1000). These results, illustrated in FIGS. 11Aand 11B, show that the E. peplus extract induced intracellular oxidationof the dye, typical of a respiratory burst. Phagocytic activity wasdetermined by a conventional method (Hudson and Hay, PracticalImmunology, 3^(rd) edition, p. 74). Cells were treated in 10% FCS-RPMI1640 at 37° C. for 20 min with introblue tetrazolium (NBT) and crude E.peplus extract (PEP001) diluted 1/1000 or 100 ng/mL TPA, followd bycounting the number of blue-stained cells in a haemocytometer. Theaverage of three fields gave figures of <2% (controls), 10% (TPA) and8.7% (E. peplus sap) cells stained blue. This demonstrates induction ofphagocytic activity, part of the normal response to infectious agents,by E. peplus sap, as shown by uptake by cells of the blue NBTprecipitate.

Experiments were also carried out using 2′,7′-dichlorofluoresceindiacetate (DCFH-DA) to measure the production of H₂O₂.(J P Robinson,Oxidative burst methods, in Handbook of Flow Cytometry Methods,Wiley-Liss Inc, pp 147-149, 1993). H₂O₂ oxidizes the non-fluorescentprobe (DCFH-DA) to a fluorescent probe that can then be detected by aflow cytometer. Peripheral blood mononuclear cells (PBMC) were extractedfrom a donor blood sample by lysis of heparinized blood and used in asuspension of 1×10⁶/mL of phosphate buffer, pH 7.3. The cells were thenincubated, with DCFH-DA (1 μL/mL of 20 mM stock) for 15 minutes to allowit to be taken up and trapped by hydrolysis with cellular esterases. Thecells were then stimulated by test compounds for 15 min at 37° C.Controls included in the experiment were unloaded control (cells with noDCFH-DA) and loaded control (cells with DCFH-DA, but no stimulation).These were used to monitor the non-specific oxidation of unstimulatedcells. The cells were then analyzed on the flow cytometer (excitation at488 nm, emission at 525±20 nm), gating each sample for individual cellpopulations—granulocytes, monocytes and lymphocytes (Table 3).

All compounds except Bryostatin induced a respiratory burst, the effectbeing strongest in granulocytes and monocytes compared with lymphocytes.Similar results were obtained by measuring the reduction, under the sameconditions, of nitroblue tetrazolium, measured as the proportion ofpurple-stained cells counted under the microscope.

Evidence for the requirement of PKC activation was obtained by additionof bisindolylmaleimide (10 μg/mL or 1 μg/mL) at the same time as PEP005,PEP006, PEP008 and PEP010. This PKC inhibitor blocked the respiratoryburst seen with TPA and PEP003.

Phagocytosis with Fluorescent Beads

Phagocytosis by peripheral blood mononuclear cells (PBMCs) was assayed(Steinkamp et al., 1982) using 1 μm Fluoresbrite™ yellow-greenfluorescent latex spheres (Polysciences, Inc., Warrington, Pa.). Asample of whole, heparinized blood was treated with drug and 5×10E7fluorescent latex beads in 10 μL of PBS added per mL of suspension.Cells were incubated and maintained in suspension for 30 min by means ofa shaker platform at 37° C. The stimulated and non-stimulated sampleswere then lysed to isolate PBMCs. The PBMCs were run on the flowcytometer measuring FITC (excitation at 488 nm, emission at 525±20 nm),gated for fluorescence (phagocytosed spheres) and light scatter (cellsize).

The data presented in Table 4 indicate that TPA, PEP006, PEP008, PEP003and PEP005 all stimulate phagocytosis in PBMCs.

EXAMPLE 10 Activation of Innate Antiviral Activity

Many viruses, including alphaviruses, are sensitive to innate antiviralactivities, which are often mediated by the activation of interferon α/βresponses (Antalis et al., 1998). Such antiviral activities inhibit theability of cells to support viral replication. For many viralinfections, including those caused by Ross River virus, viralreplication results in virus-induced cytophathic effect (CPE) or celldeath. Treatrnent of human fibroblast cells with E. peplus ingenanes wasshown to activate antiviral activity and prevented CPE induced by analphavirus infection.

Human skin fibroblasts (10e4/well) were seeded in 96 well plate and leftovernight to adhere. An extract of E. peplus ingenanes was added at 5μg/mL for 48 hr. An alphavirus (Ross River virus, T48) was then added ata dose of 1, 10 and 100 cell culture ID50 for 6 days (La Linn et al.,1996). The cytopathic effect of the viral infection was assayed usingcrystal violet staining. Protected cells stain violet, whereas cellswhich have suffered CPE detach from the plate, leaving the wellunstained. Alphavirus-induced CPE was observed in treated cells only ata 100-fold greater dose of virus than was required to induce CPE inuntreated cells, indicating that a significant degree of protection wasconferred by the E. peplus extract.

EXAMPLE 11 Protection Against Intra-Peritoneal Streptococcal Infection:Effect of PEP003 and PEP004 on Systemic Group A Streptococcal Infectionin Mice

Infection of humans with group A streptococcus (Streptococcus pyogenes)(GAS) can cause a variety of clinical manifestations including therelatively minor pharyngitis (“trep throat” and impetigo (superficialskin infection) to more severe invasive infections such as toxic shocksyndrome and necrotizing fasciitis, both of which, may lead tomultisystem organ failure. Lastly, the GAS post-infectious sequelae ofrheumatic fever (RF), rheumatic heart disease (RHD) and acuteglomerulonephritis (AGN) are a major problem in developing countries andindigenous populations, particularly in Australian Aboriginals. Currenttreatment for controlling GAS infection is with antibiotic therapy,however, since continual high dose administration of antibiotic isrequired in cases of repeated episodes of acute RF and the developmentof RHD, poor compliance is often associated with the persistence ofthese GAS-associated diseases. The development of a vaccine against GASinfection would prevent GAS-associated diseases including RF and RHD. Inthe absence of a vaccine, however, the development of new drugs withimproved anti-bacterial activity may provide promising therapeuticagents.

The inventors' aim was to test the ability of the PEP003 and PEP004 tosystemically protect against GAS infection, in vivo. Mice (n=10) weretreated with 50 μL of PEP003 (500 nM), PEP004 (1:100 dilution fromstock) or control (PBS/10% acetone), 24 hr prior to and thereafter i.p.challenge with live GAS. Two different strains of mice (Quackenbush andB10.BR) and four different GAS strains (NS-1, PL-1, 88/30 and M1) wereused. Mice were monitored for two weeks post-challenge and thepercentage survival of mice determined. Percentage survival inQuackenbush mice challenged with PL-1 GAS was 70% (PEP003), 60% (PEP004)and 40% (control) (Table 5). Control mice that had been given the samesuccessive treatment of PEP003 and PEP004 (but not challenged) to ruleout any potential adverse side effects of the compounds were then alsochallenged with PL-1; survival was 40%, 80%, and 20% for PEP003, PEP004and controls, respectively (Table 6). In the latter experiment, theprotective effect of PEP004 approached significance (p=0.06), however,small numbers of mice were used (n=5). In Quackenbush mice challengedwith NS-1 GAS, survival was 50% for PEP003 and controls, and 80% forPEP004 (Table 5). In B10.BR mice challenged with M1 GAS, survival was10% for controls, 30% for PEP003 and 0% for PEP004 (Table 5). In B10.BRmice challenged with 88/30 GAS, survival was 20% for controls, 30% forPEP004 and 0% for PEP003 (Table 5). The data indicate a possibleprotective effect of PEP004 against systemic GAS challenge inQuackenbush mice. In addition, these data indicate that a weeklytreatment regimen of PEP003 and PEP004 prior to GAS challenge may bemore effective.

EXAMPLE 12 Anti-Escherichia coli Activity of PEP003: Activation ofLeucocytes

Blood was collected into a Sodium Heparin tube (Becton DickinsonVACUTAINER) and leucocytes prepared by lysis of red blood cells(Handbook of Flow Cytometry Methods. Robinson J P. Wiley-Liss Inc 1993.Oxidative Burst Methods H₂O₂ DCF Assay by Flow cytometry p 147-149).Leucocytes were resuspended and divided equally into two tubes such thateach tube contained 7×10⁶ peripheral blood cells (PBCs). Both tubes werethen centrifuged (Beckman, GS-6) at 1000 rpm for 10 minutes. Thesupernatant was removed and the volume was then adjusted to 1 mL withRPMI 1640 (Gibco BRL, antibiotic free supplemented with 10% v/v fetalbovine serum. 100 μL of PEP003 (to give a final concentration of 23μg/mL containing 10% acetone was then added to one tube and to theother, 100 μL of PBS/10% Acetone. To each tube, 10 μL E.coli (competentcells, XL10-Blue, Stratagene) was also added (to give a ˜1/100 dilutionof a static culture). Both tubes were vortexed then centrifuged(Beckman, GS-6) at 2500 rpm for 10 minutes. Lids were loosened and thetubes were incubated at 37° C./5% CO₂. Following 16 hr incubation, thetubes were vortexed. To estimate the number of E. coli, 50 mL was takenfrom both tubes as well as the static starter culture (stored at 4° C.),transferred to Eppendorf tubes and centrifuged (Beckman, GS-15R) at10,000 rpm for 10 minutes. Supernatant (˜45 μL) was removed and thepellet resuspended in the remaining ˜5 μL. A smear was made on a glassslide using the 5 μL bacterial suspension and stained using Quick Dip(Histo.Labs, Riverstone, Australia), a modified method of theWright-Giemsa stain, which stains bacteria blue. E. coli were countedusing a conventional light microscope (×400) with an eyepiece micrometer(100 μm×100 μm). This count was then adjusted to give a total count inthe smear (area=12.5×10⁵ μm²) and expressed as the number of E. coli permL. Another method of measuring growth of E. coli was to read theabsorbance (595 nm) of the supernatant.

The results presented in FIGS. 12 and 13 show that treatment ofleucocytes with PEP003 results in a significant reduction in bacterialnumbers.

EXAMPLE 13 Treatment of Ringworm

Ringworm is a subcutaneous mycosis or dermatophytosis caused by fungi ofthe species Trichophyton, Microsporum and Epidermophyton, in which theinfection is confined to the keratinous structures of the body. A twoweek old ringworm lesion, determined to be Trichophyton mentagrophytesvar. mentagrophytes by culture, on the volar surface of the forearm ofan adult male human was treated with a single topical application ofcrude E. peplus extract and was shown to resolve after seven days.Resolution of such lesions in the absence of treatment does occur, butis considered extremely rare.

EXAMPLE 14 Treatment for Bites if Blood-sucking Insects

The bites of blood sucking insects such as mosquitos and sand fliesoften cause an itchy inflammatory reaction at the site of the bite.Although the extract mechanism of this reaction is poorly understood,mast cells and histamine release are likely components of this reaction(Greaves and Wall, 1996; Horsmanheimo et al., 1996).

In preliminary experiments, the inventors treated human sand fly biteswith E. peplus extract and found a rapid reduction in the itchysensation compared to untreated bits at a distant site. Without wishingto be bound by any proposed mechanism, the inventors believe that the E.peplus extract may strongly stimulate mast cell exocytosis and histaminerelease and thereby prevent the slow release over time of thesecompounds, a feature associated with itchiness.

EXAMPLE 15 Promoter Activation as a Means of Therapy:Effect of PEP003and PEP004 on Activation of EBV Infected Cell Lines and EBV PositiveBurkitt's Lymphoma Cell Lines

Initially the effect of PEP003 and PEP004 was tested on the B95-8 cellline (an EBV positive marmoset cell line that is used worldwide as oneof the best EBV producers). This cell line was treated with each ofthese compounds (at different concentrations) for 3 days and 7 days,respectively, and activation of EBV virus production was measured by theappearance of a viral capsid antigen (VCA) on western blots. Also, as acomparison, EBV was activated in this cell line with TPA.

To ensure that equal amounts of each sample were analyzed, the gels werestained with Coomassie blue and the loadings were adjusted to make themequal. Analyses of VCA in each of the samples showed that both PEP003and PEP004 were capable of activating EBV (at all of the concentrationsused) to similar levels as using 65 nM TPA (FIG. 14). Next the PEP003and PEP004 were assayed on two Burkitt's lymphoma cell lines and an LCL.This time only concentrations of 10⁻⁵ and 10⁻⁷ were used. Neither PEP003and PEP004 had much effect on the LCL (this LCL produces some VCAwithout and chemical induction and this was not increased by thesecompounds). PEP004 had no effect on VCA production in any of the celllines used. However, PEP003 did induce high levels of VCA in bothBurkitt's lymphoma cell lines (MutuI and BL74), but only at 10⁵concentration (FIG. 15). Similar results were obtained when the celllines were assayed for induction of BZLF1, the initial transactivator ofEBV replication (FIG. 16). The results show that PEP003 was capable ofactivating EBV in Burkitt's lymphoma cell lines, but appeared to havelittle effect on LCLs.

In conclusion, (1) both TPA and PEP003 can modulate gene expression inEBV transformed tumor cells at the doses used; (2) while PEP003 inducedVCA in MutuI cells TPA did not, indicating different modes of action;(3) surprisingly, there was no apparent effect of PEP003 onlymphoblastoid cells, indicating potential for activating latentherpesvirus in tumors without affecting the normal infection.

EXAMPLE 16 Investigation into the Effect of PEP003 on the Ability ofMelanoma Cells to Stimulate NK Activity

Melanomas and other cancers can be killed by both specific (Tcell-mediated) and non-specific (natural killer cell and othermechanisms) arms of the immune response. These killer cells can begenerated in vitro by stimulating peripheral blood T cells from selectedmelanoma patients with melanoma cells derived from the same patient(“autologous”). Natural killer cells can be recognized by their lysis ofthe natural killer-sensitive cell line K562. It has been theorized thatsome anti-tumor agents alter the susceptibility of melanomas to immuneresponses.

Peripheral blood lymphocytes from patient A02, who has a strong specificT cell response to her own melanoma cells (A02-M), were thawed andstimulated by irradiated A02-M pre-treated overnight at 37° C. with (a)PEP003 (2.25 μg/mL; 50 μM); (b) TPA (100 ng/mL); or (c) controlsolvent/buffer, and washed ×2 before addition to responding lymphocytes(washing ×2 achieves a residual agent dilution of ×100,000). After 10days of culture, the stimulated cells were harvested and used aseffectors against an NK-sensitive cell line (K562) to test for the levelof NK activity generated in culture. All determinations were performedin triplicate, at E:T ratios of 45, 15, 5 and 1.7:1. A standard 5 hour⁵¹Cr release assay was performed. Stimulations were performed in 10%fetal bovine serum/RPMI-1640.

The results presented in Table 7 and FIG. 17 indicate that pre-treatmentof melanoma cells with PEP003 significantly increases the lysis of K562compared to both TPA and the control treatment at the E:T ratio of 45:1(P<0.01 in both cases), suggesting that PEP003 increases NK activity inA02 cultures.

EXAMPLE 17 Methods for Obtaining a Low-chlorophyll, Hydrophobic FractionFrom E. peplus and Other Plant Species

Standard methods for the isolation of hydrophobic compounds from plantsinvolve alcoholic extraction of the whole plant. This produces anextract containing chlorophyll and other hydrophobic substances from theleaves that interfere with subsequent purification of compounds bysolvent extractions and chromatography. This is a particular problem inisolating highly bioactive diterpenes from members of the Euphorbiaceaefamily, due to co-migration with chlorophyll on silica gelchromatography. Two methods, both of which can be scaled up foreconomical, commercial production, have been developed to overcome thisproblem, as described in the present Example and in Example 18.

Fresh E. peplus plants (17 kg) were chopped and soaked in 150 liters ofwater at 4° C. for 20 hr. The water was pumped through 50 and 100 meshsieves, filtered through 5 and 2 micron filters and then recirculatedthrough a 100 mm diameter column of Amberlite XAD-16 (1.5 kg,conditioned successively with ethyl acetate, methanol and water) at 4°C. (approximately 1.2 L/min) for 72 hr. Adsorption of bioactivity to theresin was found to be virtually complete within 20 hr.

The resin was then washed successively with water and 50% methanol, theneluted with 1L of methanol, followed by 2×1 L acetone. The eluates wereevaporated and combined to give approximately 7 g of a thick oil. Thiswas shown by HPTLC to be substantially free of chlorophyll and tocontain the desired ingenane esters which were then purified asdescribed below.

The ability to extract diterpene esters from chopped plants in water wassurprising given their relative hydrophobicity and water insolubility. Avariety of manual (cutting with scissors) and mechanical (rotarycutters, motor-driven mulcher) plant maceration methods were successful,as was extraction at room temperature. Adsorption to the XAD-16 could beachieved by stirring the resin with the filtered or unfiltered waterextract and then pouring off the latter. Filtration could also becarried out with minimal loss of bioactivity using diatomaceous earth,or membrane filters (220-650 microns). XAD-7 and XAD-4 were as effectiveas XAD-16.

The hydrophobic adsorbent polyamide (ICN Biomedical Research Products)was also used to trap the diterpenes from water; it had the advantage ofallowing the diterpene esters to be selectively eluted with 50-80%methanol, thus separating them from inactive, hydrophobic compounds,which remained on the column.

EXAMPLE 18 Method for Separation of Ingenane Esters From OtherDiterpenes

The following method is based upon the surprising discovery that thestems of E. peplus contain approximately 90% of the bioactive diterpenesand significantly less chlorophyll compared with the leaves.

The plants are dried in air, shaken to remove the leaves and the stemscompressed and covered with an equal weight of methanol for 24 hr. Thesolvent is then poured off, evaporated to dryness under reduced pressureand the residue dissolved in methanol for chromatography on SephadexHL20 as described below. This method is also suitable for isolation oflow-chlorophyll fractions from other plant species.

A solution of crude methanol extract from E. peplus in 4 mL 90% ethanolwas loaded onto a 25 mm×1000 mm column and eluted with 90% methanol.Fractions (4 mL) were analysed by HPTLC (silica gel, developed with 4:1toluene: acetone and heated with phosphoric acid at 110 degrees for 15min). Typically, fractions 54-63 contained jatrophane and pepluaneesters and fractions 64-77 the ingenane esters, thus achievingsatisfactory separation. Bioactivity, as judged by induction of bipolarmorphology in the human melanoma cell line MM96L, was retained, as forexample disclosed in PCT/AU98/00656.

This separation was surprising because the polarity of the ingenaneesters as judged by HPTLC on silica completely overlapped the rangeshown by the jatrophane and pepluane esters.

EXAMPLE 19 Process for the Purification of Diterpene Esters From E.peplus

Crude extracts obtained by the methods according to Examples 17 or 18above, or by ether extraction of latex, were fractionated by SephadexHL-20 chromatography (as above). Appropriate fractions from the latterwere combined, the methanol evaporated under reduced pressure and theremaining water removed by freeze-drying or by ether extraction. Thissample (200 μL of 100 mg/mL in methanol per injection) was fractionatedby HPLC on a Phenomenex Luna 250×10 mm C18 column with a Phenomenexguard column in 70-100% methanol at 2 mL/min, with detection at 230 nm.Jatrophane and pepluane esters appeared at 25-42 min, PEP005 at 42-44min, PEP008 at 46-50 min, and PEP006 at 50-54 min. Similar types ofseparation have been obtained by HPLC on C3 and C8 columns.

Fractions pooled from repeated runs were evaporated to dryness (rotaryevaporater or freeze dryer), and stored in acetone at −20° C. underargon or nitrogen.

EXAMPLE 20 Activation of Leukocytes by Diterpene Esters, for SelectiveKilling of Human Tumor Cells in Culture

Leukocytes obtained by lysis of human peripheral blood were added to5000 MM96L human melanoma cells or 7000 neonatal foreskin fibroblastsper microtitre well at effector: target ratios of 1000, 100 and 10:1.Ing9 (60 ng/mL) was added and after 48 hr incubation the cultures werewashed and labelled with [3H]-thymidine for 2 hr. At 100:1 ratio ofeffector:target cells, the melanoma cells showed 9% survival with PEP008whereas the normal fibroblasts had 100% survival. Untreated leukocyteshad no effect on cell survival.

These results indicate that the diterpene esters of the inventionactivate human peripheral blood leukocytes to produce, in aPKC-dependent manner, phagocytosis and a respiratory burst which arepotentially lethal to micro-organisms and other cells.

This example shows that drug-activated, PKC-dependent processes candirect tumor-specific killing by cells of the innate immune system.

EXAMPLE 21 Pretreatment of Human Tumor Cells in Culture with DiterpeneEsters Potentiates Selective Killing by Untreated Leukocytes

The question of whether drug treatment of the target tumor cells causesthem to become susceptible to effector cells of the immune system wasaddressed as follows.

Leukocytes obtained by lysis of human peripheral blood were added to5000 MM96L human melanoma cells or 7000 neonatal foreskin fibroblastsper microtitre well at effector: target ratios of 1000, 100 and 10:1.The target cells had been treated with 60 ng/mL PEP008 for 20 hrbeforehand, and washed and the medium replaced before the leukocyteswere added. After 48 hr incubation with the leukocytes the cultures werewashed and labelled with [3H]-thymidine for 2 hr. At 100:1 ratio ofeffector:target cells, the melanoma cells showed 12% survival withPEP008 whereas the normal fibroblasts had 100% survival. Untreatedleukocytes had no effect on cell survival.

This result showed that the drugs also act by making tumor cellsspecifically sensitive to lysis by the immune system.

EXAMPLE 22 Topical Composition A for the Treatment of ConditionsAffecting Skin (e.g. Infections, Skin Cancers)

Tinctures: Compounds of the invention were diluted into, acetone,ethanol or isopropanol to the same final bioactivity as the E. pepluslatex as measured by bipolar activity in MM96L human melanoma cells (10million bp units per mL). Samples (2-5 μL) were applied daily for 3 daysto the surface of mouse melanoma B16 tumor 3-5 days after implantings.c. 1 million cells on the flanks of nude mice. Efficacy, defined as67% or more sites cured, was obtained for E. peplus sap, PEP005, PEP008and a mixture of PEP005, PEP006 and PEP008.

EXAMPLE 23 Topical Composition B for the Treatment of ConditionsAffecting Skin (e.g. Infections, Skin Cancers)

Creams and gels: A variety of hydrophobic cream bases was found to beineffective when used to deliver compounds to the skin as describedabove for the tinctures. Efficacy was obtained with the use of anisopropanol gel, formulated as described for the tinctures.

The results show that E. peplus sap and its terpenoid componentsactivate PKC, with consequent potential to induce a wide range ofcellular responses without the high tumor promoting activity of TPA. Thecarboxypeptidase activity may have application in enhancement of tissuepenetration and in antigen processing for optimal immune responses.

Overall, the results indicate that E. peplus extract induces a set ofcellular responses with affects PKC, cell cycle genes and inflammatorymediators, some but by no means all of which are similar to the actionof TPA. In particular, the results indicate that E. peplus sap and itsterpenoid components are useful in the treatment of a variety ofinfections and as adjuvants for stimulating immune responses.

EXAMPLE 24 Effect of Saps Derived from Other Members of theEuphorbiaceae Family on MM96L Cells

Sap was collected from Synadenium grantii, Synadenium compactum,Mondenium lugardae, Mondenium guentheri, Endadenium gossweileni, and E.peplus and serially diluted ten-fold up to 10⁻⁷ into sterile 1.5 mLEppendorf™ tubes using growth medium. Ten-microliter aliquots of eachdilution, in the presence or absence of the PKC inhibitorbisindolylmaleimide (1 μg/mL or 10 μg/mL), were added to 5000 MM96Lcells per well of a microtitre plate. After 3 days, cells were examinedfor cytotoxicity or differentiation to a bipolar dendritic phenotype.

The results presented in Table 8 show that the saps of S. grantii, S.compactum, M. lugardae, M. guentheri, and E. gossweileni, like that ofE. peplus, induce the differentiation of MM96L cells to a bipolarphenotype and that this differentiation is inhibited by thebisindolylmaleimide. This inhibition strongly suggests that the activecomponents of the saps induce cell differentiation by inhibition of PKCactivity. The results also show that at higher concentrations (10⁻⁴ andabove), the saps are effective in killing MM96L cells.

EXAMPLE 25 Effect of Saps Derived from Other Members of theEuphorbiaceae Family on JAM Cells

The saps of Example 24 were also examined for their cytotoxic effect onthe ovarian carcinoma cell line JAM. Ten-microliter aliquots of eachdilution of sap, prepared according to Example 24 in the presence orabsence of the PKC inhibitor bisindolylmaleimide (10 μg/mL), or in thepresence or absence of the PKC phorbol ester binding site ligand phorboldibutyrate, were added to 5000 JAM cells per well of a microtitre plate.After three days, the cells were fixed with ethanol and the number ofcells compared with untreated controls stained with sulfurhodamine B.

The results presented in FIGS. 18A and 18B indicate that, like the sapof E. peplus, the saps of S. grantii, S. compactum, M. Iugardae, M.guentheri, and E. gossweileni, at concentrations of 10⁻⁴ and above, areeffective in killing JAM cells. These results also show thatcytotoxicity is inhibited by bisindolylmaleimide, suggesting that thiseffect is mediated by modulation of PKC.

Inspection of FIG. 18C reveals that the cytotoxic effects of sapsderived from M. guentheri and E. gossweileni were blocked in thepresence of phorbol dibutyrate, suggesting that the active components ofthese saps mediate their cytotoxicity by binding to the phorbol esterbinding site of PKC.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

BIBLIOGRAPHY

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1. A method for effecting the recognition in a host of a latent virusinfection, which method comprises administering to said host an amountof a chemical agent effective to activate said virus to cause the virusto replicate itself and, become visible to the host, said chemical agentbeing an isolated ingenane, or pharmaceutically acceptable salt thereof,said ingenane having the formula:

wherein: R₂₄, R₂₅ and R₂₆ are independently selected from hydrogen, R₂₇,R₂₈, F, Cl, Br, I, OH, CN, OR₂₇, SR₂₇, NR₂₇R₂₈, N(═O)₂, NR₂₇OR₂₈,ONR₂₇R₂₈, SOR₂₇, SO₂R₂₇, SO₃R₂₇, SONR₂₇R₂₈, SO₂NR₂₇R₂₈, SO₃NR₂₇R₂₈,P(R₂₇)₃, P(═O)(R₂₇)₃, Si(R₂₇)₃, B(R₂₇)₂, (C═X)R₂₉ or X(C═X)R₂₉ where Xis selected from sulfur, oxygen and nitrogen; R₂₇ and R₂₈ are eachindependently selected from C₁-C₂₀ alkyl (branched and/or straightchained), aryl C₁-C₂₀ alkyl, C₃-C₈ cycloalkyl, C₆-C₁₄ aryl, C₁-C₁₄heteroaryl, C₁-C₁₄ heterocycle, C₂-C₁₀ alkenyl (branched and/or straightchained), C₂-C₁₀ alkynyl (branched and/or straight chained), C₁-C₁₀heteroarylalkyl, C₁-C₁₀ alkoxyalkyl, C₁-C₁₀ haloalkyl, dihaloalkyl,trihaloalkyl, haloalkoxy, C₁-C₁₀ alkyl which is unsubstituted orsubstituted by CN, OR₃₀, SR₃₀, NR₃₀R₃₁, N═(O)₂, NR₃₀OR₃₁, ONR₃₀R₃₁,SOR₃₀, SO₂R₃₀SO₃R₃₀, SONR₃₀R₃₁, SO₂NR₃₀R₃₁, SO₃NR₃₀R₃₁, P(R₃₀)₃,P═(O)(R₃₀)₃, Si(R₃₀)₃, and B(R₃₀)₂; R₂₉ is selected from R₂₇, R₂₈, CN,COR₂₇, CO₂R₂₇, OR₂₇, SR₂₇, NR₂₇R₂₈, N(═O)₂, NR₂₇OR₂₈, ONR₂₇R₂₈, SOR₂₇,SO₂R₂₇, SO₃R₂₇, SONR₂₇R₂₈, SO₂NR₂₇R₂₈, SO₃NR₂₇R₂₈, P(R₂₇)₃, P(═O)(R₂₇)₃,Si(R₂₇)₃, and B(R₂₇)₂ SO₃NR₂₇R₂₈, P(R₂₇)₃, P(═O)(R₂₇)₃, Si(R₂₇)₃, andB(R₂₇)₂; and each R₃₀ and R₃₁ are independently C₁-C₂₀ alkyl, arylC₁-C₂₀ alkyl, C₃-C₈ cycloalkyl, C₆-C₁₄ aryl, C₁-C₁₄ heteroaryl,C₁-C₁₄-heterocycle, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀heteroarylalkyl, C₁-C₁₀ alkoxyalkyl, C₁-C₁₀ haloalkyl, dihaloalkyl,trihaloalkyl, and haloalkoxy.
 2. The method according to claim 1 whereinR₂₄ is H.
 3. The method according to claim 1 wherein R₂₄ is OAcetyl. 4.A method according to claim 1 wherein R₂₄ is OH.
 5. A method accordingto claim 1 wherein R₂₅ and R₂₆ are OH.
 6. The method according to claim1 wherein said chemical agent is 20-O-acetyl-ingenol-3-angelate or anester derivative thereof represented by Formula VI or a pharmaceuticallyacceptable salt of these.
 7. The method according to claim 1 whereinsaid chemical agent is ingenol-3-angelate, or an ester derivativethereof represented by Formula VI or a pharmaceutically acceptable saltof these.
 8. The method according to claim 1 wherein said chemical agentis 20-deoxy-ingenol-3-angelate or an ester derivative thereofrepresented by Formula VI or a pharmaceutically acceptable salt ofthese.
 9. The method according to any one of claims 6, 7 or 8, whereinsaid ester derivative is an acetylated derivative.
 10. The methodaccording to claim 1 wherein said subject is human.
 11. The methodaccording to claim 1 wherein R₂₅ and R₂₆ are OH and R₂₄ is hydrogen,O-Acetyl or OH.
 12. The method according to claim 1 wherein saidchemical agent is ingenol-3-angelate.
 13. The method according to claim1 wherein the chemical agent is obtainable from a species of Euphorbia.14. The method according to claim 1 wherein an anti-viral agent isadditionally administered to the subject.
 15. The method according toclaim 14 wherein said anti-viral agent is administered simultaneously orsequentially with or separately from the chemical agent.
 16. The methodaccording to claim 13 wherein the species of Euphorbia is selected fromEuphorbia aaron-rossii, Euphorbia abbreviata, Euphorbia acuta, Euphorbiaalatocaulis, Euphorbia albicaulis, Euphorbia algomarginata, Euphorbiaaliceae, Euphorbia alta, Euphorbia anacampseros, Euphorbia andromedae,Euphorbia angusta, Euphorbia anthonyi, Euphorbia antiguensis, Euphorbiaapocynifolia, Euphorbia arabica, Euphorbia arundelana, Euphorbiaastroites, Euphorbia atrococca, Euphorbia baselicis, Euphorbiabatabanensis, Euphorbia bergenri, Euphorbia bermudiana, Euphorbiabicolor, Euphorbia biformis, Euphorbia bifurcata, Euphorbia bilobata,Euphorbia biramensis, Euphorbia biuncialis, Euphorbia blepharostipula,Euphorbia blodgetti, Euphorbia boerhaavioides, Euphorbia boliviana,Euphorbia bracei, Euphorbia brachiata, Euphorbia brachycera, Euphorbiabrandegee, Euphorbia brittonii, Euphorbia caesia, Euphorbia calcicola,Euphorbia campestris, Euphorbia candelabrum, Euphorbia capitellata,Euphorbia carmenensis, Euphorbia carunculata, Euphorbia cayensis,Euphorbia celastroides, Euphorbia chalicophila, Euphorbia chamaerrhodos,Euphorbia chamaesula, Euphorbia chiapensis, Euphorbia chiogenoides,Euphorbia cinerascens, Euphorbia clarionensis, Euphorbia colimae,Euphorbia colorata, Euphorbia commutata, Euphorbia consoquitlae,Euphorbia convolvuloides, Euphorbia corallifera, Euphorbia creberrima,Euphorbia crenulata, Euphorbia cubensis, Euphorbia cuspidata, Euphorbiacymbiformis, Euphorbia darlingtonii, Euphorbia defoliata, Euphorbiadegeneri, Euphorbia deltoidea, Euphorbia dentata, Euphorbia depressaEuphorbia dictlyosperma, Euphorbia dictyosperma, Euphorbia dioeca,Euphorbia discoidalis, Euphorbia dorsiventralis, Euphorbia drumondii,Euphorbia duclouxii, Euphorbia dussii, Euphorbia eanophylla, Euphorbiaeggersii, Euphorbia eglandulosa, Euphorbia elata, Euphorbia enalla,Euphorbia eriogonoides, Euphorbia eriophylla, Euphorbia esculaeformis,Euphorbia espirituensis, Euphorbia esula, Euphorbia excisa, Euphorbiaexclusa, Euphorbia exstzipitata, Euphorbia exstipulata, Euphorbiafendleri, Euphorbia filicaulis, Euphorbia filiformis, Euphorbia florida,Euphorbia fruticulosa, Euphorbia garber, Euphorbia gaumerii, Euphorbiagerardiana, Euphorbia geyeri, Euphorbia glyptosperma, Euphorbiagorgonis, Euphorbia gracilior, Euphorbia gracillima, Euphorbia gradyi,Euphorbia graminea, Euphorbia graminiea Euphorbia grisea, Euphorbiaguadalajarana, Euphorbia guanarensis, Euphorbia gymnadenia, Euphorbiahaematantha, Euphorbia hedyotoides, Euphorbia heldrichii, Euphorbiahelenae, Euphorbia helleri, Euphorbia helwigii, Euphorbia henricksonii,Euphorbia heterophylla, Euphorbia hexagona, Euphorbia hexagonoides,Euphorbia hinkleyorum, Euphorbia hintonii, Euphorbia hirtula, Euphorbiahirta, Euphorbia hooveri, Euphorbia humistrata, Euphorbia hypericifolia,Euphorbia inundata, Euphorbia involuta, Euphorbia jaliscensis, Euphorbiajejuna, Euphorbia johnston, Euphorbia juttae, Euphorbia knuthii,Euphorbia lasiocarpa, Euphorbia lata, Euphorbia latazi, Euphorbialatericolor, Euphorbia laxiflora Euphorbia lecheoides, Euphorbialedienii, Euphorbia leucophylla, Euphorbia lineata, Euphorbialinguiformis, Euphorbia longecornuta, Euphorbia longepetiolata,Euphorbia longeramosa, Euphorbia longinsulicola, Euphorbia longipila,Euphorbia lupulina, Euphorbia lurida, Euphorbia lycicides, Euphorbiamacropodoides, macvaughiana, Euphorbia manca, Euphorbia mandoniana,Euphorbia mangleti, Euphorbia mango, Euphorbia marylandica, Euphorbiamayana, Euphorbia melanadenia, Euphorbia melanocarpa, Euphorbiameridensis, Euphorbia mertonii, Euphorbia mexiae, Euphorbiamicrocephala, Euphorbia microclada, Euphorbia micromera, Euphorbiamisella, Euphorbia missurica, Euphorbia montana, Euphorbia montereyana,Euphorbia multicaulis, Euphorbia multiformis, Euphorbia multinodis,Euphorbia multiseta, Euphorbia muscicola, Euphorbia neomexicana,Euphorbia nephradenia, Euphorbia niqueroana, Euphorbia oaxacana,Euphorbia occidentalis, Euphorbia odontodenia, Euphorbia olivacea,Euphorbia olowaluana, Euphorbia opthalmica, Euphorbia ovata, Euphorbiapachypoda, Euphorbia pachyrhiza, Euphorbia padifolia, Euphorbia palmeri,Euphorbia paludicola, Euphorbia parciflora, Euphorbia parishii,Euphorbia parryi, Euphorbia paxiana, Euphorbia pediculifera, Euphorbiapeplidion, Euphorbia peploides, Euphorbia peplus, Euphorbia pergamena,Euphorbia perlignea, Euphorbia petaloidea, Euphorbia petaloidea,Euphorbia petrina, Euphorbia picachensis, Euphorbia pilosula, Euphorbiapilulifera, Euphorbia pinariona, Euphorbia pinetorum, Euphorbiapionosperma, Euphorbia platysperma, Euphorbia plicata, Euphorbiapoeppigii, Euphorbia poliosperma, Euphorbia polycarpa, Euphorbiapolycnemoides, Euphorbia polyphylla, Euphorbia portoricensis, Euphorbiaportulacoides Euphorbia portulana, Euphorbia preslii, Euphorbiaprostrata, Euphorbia pteroneura, Euphorbia pycnanthema, Euphorbiaramosa, Euphorbia rapulum, Euphorbia remyi, Euphorbia retroscabra,Euphorbia revoluta, Euphorbia rivularis, Euphorbia robusta, Euphorbiaromosa, Euphorbia rubida, Euphorbia rubrosperma, Euphorbia rupicola,Euphorbia sanmartensis, Euphorbia saxatilis M. Bieb, Euphorbiaschizoloba, Euphorbia sclerocyathium, Euphorbia scoputorum, Euphorbiasenilis, Euphorbia serpyllifolia, Euphorbia serrula, Euphorbia setilobaEngeim, Euphorbia sonorae, Euphorbia soobyi, Euphorbia sparsiflora,Euphorbia sphaerosperma, Euphorbia syphilitica, Euphorbia spruceana,Euphorbia subcoerulea, Euphorbia stellata, Euphorbia submammilaris,Euphorbia subpeltata, Euphorbia subpubens, Euphorbia subreniforme,Euphorbia subtrfoliata, Euphorbia succedanea, Euphorbia tamaulipasana,Euphorbia telephioides, Euphorbia tenuissima, Euphorbia tetrapora,Euphorbia tirucalli, Euphorbia tomentella, Euphorbia tomentosa,Euphorbia torralbasii, Euphorbia tovariensis, Euphorbia trachysperma,Euphorbia tricolor, Euphorbia troyana, Euphorbia tuerckheimii, Euphorbiaturczaninowii, Euphorbia umbellulata, Euphorbia undulata, Euphorbiavermiformis, Euphorbia versicolor, Euphorbia villifera, Euphorbiaviolacea, Euphorbia whitei, Euphorbia xanti Engeim, Euphorbia xylopodaGreenm., Euphorbia yayalesia Urb., Euphorbia yungasensis, Euphorbiazeravschanica and Euphorbia zinniiflora.
 17. The method according toclaim 13 wherein the species of Euphorbia is Euphorbia peplus.